Role of chemokines and their receptors in viral persistence and liver damage during chronic hepatitis C virus infection

Chemokines produced in the liver during hepatitis C virus(HCV) infection induce migration of activated T cells from the periphery to infected parenchyma.The milieu of chemokines secreted by infected hepatocytes is predominantly associated with the T-helper cell/Tc1 T cell(Th1/Tc1) response.These chemokines consist of CCL3(macrophage inflammatory protein-1α;MIP-1α),CCL4(MIP-1β),CCL5(regulated on activation normal T cell expressed and secreted;RANTES),CXCL10(interferon-γ-inducible protein-10;IP-10),CXCL11(interferon-inducible T-cell α chemoattractant;I-TAC),and CXCL9(monokine induced by interferon γ;Mig) and they recruit T cells expressing either CCR5 or CXCR3 chemokine receptors.Intrahepatic and peripheral blood levels of these chemokines are increased during chronic hepatitis C.The interaction between chemokines and their receptors is essential in recruiting HCV-specific T cells to control the infection.When the adaptive immune response fails in this task,non-specific T cells without the capacity to control the infection are also recruited to the liver,and these are ultimately responsible for the persistent hepatic damage.The modulation of chemokine receptor expression and chemokine secretion could be a viral escape mechanism to avoid specific T cell migration to the liver during the early phase of infection,and to maintain liver viability during the chronic phase,by impairing non-specific T cell migration.Some chemokines and their receptors correlate with liver damage,and CXCL10(IP-10) and CXCR3 levels have shown a clinical utility as predictors of treatment response outcome.The regulation of chemokines and their receptors could be a future potential therapeutic target to decrease liver inflammation and to increase specific T cell migration to the infected liver.

Acquired amegakaryocytic thrombocytopenia previously diagnosed as idiopathic thrombocytopenic purpura in a patient with hepatitis C virus infection

We report the first case of a patient with hepatitis C virus(HCV) infection and idiopathic thrombocytopenic purpura(ITP), who later developed acquired amegakaryocytic thrombocytopenia(AAMT), with autoantibodies to the thrombopoietin(TPO) receptor(c-Mpl). A 64-year-old woman, with chronic hepatitis C, developed severe thrombocytopenia and was diagnosed with ITP. She died of liver failure. Autopsy revealed cirrhosis and liver carcinoma. In the bone marrow, a marked reduction in the number of megakaryocytes was observed, while other cell lineages were preserved. Therefore, she was diagnosed with AAMT. Additionally, autoantibodies to c-Mpl were detected in her serum. Autoantibodies to c-Mpl are one of the causes of AAMT, acting through inhibition of TPO function, megakaryocytic maturation, and platelet formation. HCV infection induces several autoantibodies. HCV infection might also induce autoantibodies to c-Mpl, resulting in the development of AAMT. This mechanism may be one of the causes of thrombocytopenia in patients with HCV infection.

Hepatitis C virus core protein modulates several signaling pathways involved in hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is the fifth most common cancer, and hepatitis C virus(HCV) infection plays a major role in HCC development. The molecular mechanisms by which HCV infection leads to HCC are varied. HCV core protein is an important risk factor in HCV-associated liver pathogenesis and can modulate several signaling pathways involved in cell cycle regulation, cell growth promotion, cell proliferation, apoptosis, oxidative stress and lipid metabolism. The dysregulation of signaling pathways such as transforming growth factor β(TGF-β), vascular endothelial growth factor(VEGF), Wnt/β-catenin(WNT), cyclooxygenase-2(COX-2) and peroxisome proliferator-activated receptor α(PPARα) by HCV core protein is implicated in the development of HCC. Therefore, it has been suggested that this protein be considered a favorable target for further studies in the development of HCC. In addition, considering the axial role of these signaling pathways in HCC, they are considered druggable targets for cancer therapy. Therefore, using strategies to limit the dysregulation effects of core protein on these signaling pathways seems necessary to prevent HCV-related HCC.

How hepatitis C virus invades hepatocytes: The mystery of viral entry

Hepatitis C virus(HCV)infection is a global health problem,with an estimated 170 million people being chronically infected.HCV cell entry is a complex multi-step process,involving several cellular factors that trigger virus uptake into the hepatocytes.The high-density lipoprotein receptor scavenger receptor class B type I,tetraspanin CD81,tight junction protein claudin-1,and occludin are the main receptors that mediate the initial step of HCV infection.In addition,the virus uses cell receptor tyrosine kinases as entry regulators,such as epidermal growth factor receptor and ephrin receptor A2.This review summarizes the current understanding about how cell surface molecules are involved in HCV attachment,internalization,and membrane fusion,and how host cell kinases regulate virus entry.The advances of the potential antiviral agents targeting this process are introduced.

Interaction of hepatitis C virus envelope glycoprotein E2 with the large extracellular loop of tupaia CD81

AIM: To further analyze the interaction of tupaia CD81 with hepatitis C virus (HCV) envelope protein E2. METHODS: A tupaia CD81 large extracellular loop (CD81 LEL), which binds to HCV E2 protein, was cloned and expressed as a GST-fusion protein, and interaction of HCV E2 protein with a tupaia CD81 LEL was evaluated by enzyme-linked immunosorbent assay (EIA). RESULTS: Although tupaia and human CD81 LEL differed in 6 amino acid changes, tupaia CD81 LEL was strongly recognized by anti-CD81 antibodies against human CD81 LEL conformation-dependent epitopes. Investigating LEL CD81-E2 interactions by EIA, we demonstrated that binding of tupaia CD81 LEL GST fusion protein to recombinant HCV E2 protein was markedly reduced compared to binding of human CD81 LEL GST fusion protein to recombinant HCV E2 protein. CONCLUSION: These data suggest that the structural differences in-between the tupaia and human CD81 may alter the interaction of the large extracellular loop with HCV envelope glycoprotein E2. These findings may be important for the understanding of the mechanisms of binding and entry of HCV to PTHs.

Hepatitis C virus infection and insulin resistance

Approximately 170 million people worldwide are chronically infected with hepatitis C virus(HCV).Chronic HCV infection is the leading cause for the development of liver fibrosis,cirrhosis,hepatocellular carcinoma(HCC)and is the primary cause for liver transplantation in the western world.Insulin resistance is one of the pathological features in patients with HCV infection and often leads to development of typeⅡdiabetes.Insulin resistance plays an important role in the development of various complications associated with HCV infection.Recent evidence indicates that HCV associated insulin resistance may result in hepatic fibrosis,steatosis,HCC and resistance to anti-viral treatment.Thus,HCV associated insulin resistance is a therapeutic target at any stage of HCV infection.HCV modulates normal cellular gene expression and interferes with the insulin signaling pathway.Various mechanisms have been proposed in regard to HCV mediated insulin resistance,involving up regulation of inflammatory cytokines,like tumor necrosis factor-α,phosphorylation of insulin-receptor substrate-1,Akt,up-regulation of gluconeogenic genes like glucose 6 phosphatase,phosphoenolpyruvate carboxykinase 2,and accumulation of lipid droplets.In this review,we summarize the available information on how HCV infection interferes with insulin signaling pathways resulting in insulin resistance.

Inhibitory effect of miR-125b on hepatitis C virus core protein-induced TLR2/My D88 signaling in THP-1 cells

AIM: To investigate the role of mi R-125 b in regulating monocyte immune responses induced by hepatitis C virus(HCV) core protein.METHODS: Monocytic THP-1 cells were treated with various concentrations of recombinant HCV core protein, and cytokines and mi R-125 b expression in these cells were analyzed. The requirement of Tolllike receptor 2(TLR2) or My D88 gene for HCV core protein-induced immune responses was determined by the transfection of THP-1 cells with gene knockdown vectors expressing either TLR2 si RNA or My D88 si RNA. The effect of mi R-125 b overexpression on TLR2/My D88 signaling was examined by transfecting THP-1 cells with mi R-125 b mimic RNA oligos.RESULTS: In response to HCV core protein stimulation, cytokine production was up-regulated and mi R-125 b expression was down-regulated in THP-1 cells. The modulatory effect of HCV core protein on cellular events was dose-dependent and required functional TLR2 or My D88 gene. Forced mi R-125 b expression abolished the HCV core protein-induced enhancement of tumor necrosis factor-α, interleukin(IL)-6, and IL-10 expression by 66%, 54%, and 66%, respectively(P < 0.001), by inhibiting My D88-mediated signaling, including phosphorylation of NF-k Bp65, ERK, and P38.CONCLUSION: The inverse correlation between mi R-125 b and cytokine expression after HCV core challenge suggests that mi R-125 b may negatively regulate HCVinduced immune responses by targeting TLR2/My D88 signaling in monocytes.

Hepatitis C virus core protein-induced miR-93-5p upregulation inhibits interferon signaling pathway by targeting IFNAR1

AIM To investigate the mechanism by which hepatitis C virus(HCV) core protein-induced mi R-93-5 p up-regulation regulates the interferon(IFN) signaling pathway.METHODS HCV-1 b core protein was exogenously expressed in Huh7 cells using pc DNA3.1(+) vector. The expression of mi R-93-5 p and interferon receptor 1(IFNAR1) was measured using quantitative reverse transcriptionpolymerase chain reaction and Western blot. The protein expression and phosphorylation level of STAT1 were evaluated by Western blot. The overexpression and silencing of mi R-93-5 p and IFNAR1 were performed using mi R-93-5 p agomir and antagomir, and pc DNA3.1-IFNAR1 and IFNAR1 si RNA, respectively. Luciferase assay was used to identify whether IFNAR1 is a target of mi R-93-5 p. Cellular experiments were also conducted.RESULTS Serum mi R-93-5 p level was increased in patients with HCV-1 b infection and decreased to normal level after HCV-1 b clearance, but persistently increased in those with pegylated interferon-α resistance, compared with healthy subjects. Serum mi R-93-5 p expression had an AUC value of 0.8359 in distinguishing patients with pegylated interferon-α resistance from those with pegylated interferon-α sensitivity. HCV-1 b core protein increased mi R-93-5 p expression and induced inactivation of the IFN signaling pathway in Huh7 cells. Furthermore, IFNAR1 was identified as a direct target of mi R-93-5 p, and IFNAR1 restore could rescue mi R-93-5 p-reduced STAT1 phosphorylation, suggesting that the mi R-93-5 p-IFNAR1 axis regulates the IFN signaling pathway.CONCLUSION HCV-1 b core protein-induced mi R-93-5 p up-regulation inhibits the IFN signaling pathway by directly targeting IFNAR1, and the mi R-93-5 p-IFNAR1 axis regulates STAT1 phosphorylation. This axis may be a potential therapeutic target for HCV-1 b infection.

(-)-Epigallocatechin-3-gallate enhances poly I:C-induced interferon-λ1 production and inhibits hepatitis C virus replication in hepatocytes

AIM To investigate the effect of(-)-epigallocatechin-3-gallate(EGCG) on polyinosinic-polycytidylic acid(poly I:C)-triggered intracellular innate immunity against hepatitis C virus(HCV) in hepatocytes. METHODS A cell culture model of HCV infection was generated by infecting a hepatoma cell line, Huh7, with HCV JFH-1 strain(JFH-1-Huh7). Poly I:C with a high molecular weight and EGCG were used to stimulate the JFH-1-Huh7 cells. Real-time reverse transcription-polymerase chain reaction was used to detect the expression levels of intracellular m RNAs and of intracellular and extracellular HCV RNA. Enzyme-linked immunosorbent assay was used to evaluate the interferon(IFN)-λ1 protein level in the cell culture supernatant. Immunostaining was used to examine HCV core protein expression in Huh7 cells.RESULTS Our recent study showed that HCV replication could impair poly I:C-triggered intracellular innate immune responses in hepatocytes. In the current study, we showed that EGCG treatment significantly increased the poly I:C-induced expression of Toll-like receptor 3(TLR3), retinoic acid-inducible gene I, and IFN-λ1 in JFH-1-Huh7 cells. In addition, supplementation with EGCG increased the poly I:C-mediated antiviral activity in JFH-1-Huh7 cells at the intracellular and extracellular HCV RNA and protein levels. Further investigation of the mechanisms showed that EGCG treatment significantly enhanced the poly I:C-induced expression of IFN-regulatory factor 9 and several antiviral IFNstimulated genes, including ISG15, ISG56, myxovirus resistance A, and 2'-5'-oligoadenylate synthetase 1, which encode the key antiviral elements in the IFN signaling pathway. CONCLUSION Our observations provide experimental evidence that EGCG has the ability to enhance poly I:C-induced intracellular antiviral innate immunity against HCV replication in hepatocytes.

MicroRNA-185-5p mediates regulation of SREBP2 expression by hepatitis C virus core protein

AIM: To investigate the molecular mechanism for regulation of cholesterol metabolism by hepatitis C virus(HCV) core protein in Hep G2 cells.METHODS: HCV genotype 1b core protein was cloned and expressed in Hep G2 cells. The cholesterol content was determined after transfection. The expression of sterol regulatory element binding protein 2(SREBP2) and the rate-limiting enzyme in cholesterol synthesis(HMGCR) was measured by quantitative real-time PCR and immunoblotting after transfection. The effects of core protein on the SREBP2 promoter and 3'-untranslated region were analyzed by luciferase assay. We used different target predictive algorithms, micro RNA(mi RNA) mimics/inhibitors, and site-directed mutation to identify a putative target of a particular mi RNA.RESULTS: HCV core protein expression in Hep G2 cells increased the total intracellular cholesterol level(4.05 ± 0.17 vs 6.47 ± 0.68, P = 0.001), and this increase corresponded to an increase in SREBP2 and HMGCR m RNA levels(P = 0.009 and 0.037, respectively) and protein expression. The molecular mechanism studyrevealed that the HCV core protein increased the expression of SREBP2 by enhancing its promoter activity(P = 0.004). In addition, mi R-185-5p expression was tightly regulated by the HCV core protein(P = 0.041). Moreover, overexpression of mi R-185-5p repressed the SREBP2 m RNA level(P = 0.022) and protein expression. In contrast, inhibition of mi R-185-5p caused upregulation of SREBP2 protein expression. mi R-185-5p was involved in the regulation of SREBP2 expression by HCV core protein. CONCLUSION: HCV core protein disturbs the cholesterol homeostasis in Hep G2 cells via the SREBP2 pathway; mi R-185-5p is involved in the regulation of SREBP2 by the core protein.

CD36 genetic variation, fat intake and liver fibrosis in chronic hepatitis C virus infection

AIM To analyze the association of the CD36 polymorphism(rs1761667) with dietary intake and liver fibrosis(LF) in chronic hepatitis C(CHC) patients. METHODS In this study, 73 patients with CHC were recruited. The CD36 genotype(G > A) was determined by a TaqM an real-time PCR system. Dietary assessment was carried out using a three-day food record to register the daily intake of macronutrients. Serum lipids and liver enzymes were measured by a dry chemistry assay. LF evaluated by transient elastography(Fibroscan~)and APRI score was classified as mild LF(F1-F2) and advanced LF(F3-F4).RESULTS Overall, the CD36 genotypic frequencies were AA(30.1%), AG(54.8%), and GG(15.1%), whereas the allelic A and G frequencies were 57.5% and 42.5%, respectively. CHC patients who were carriers of the CD36 AA genotype had a higher intake of calories attributable to total fat and saturated fatty acids than those with the non-AA genotypes. Additionally, aspartate aminotransferase(AST) serum values were higher in AA genotype carriers compared to non-AA carriers(91.7 IU/L vs 69.8 IU/L, P = 0.02). Moreover, the AA genotype was associated with an increase of 30.23 IU/L of AST(β = 30.23, 95%CI: 9.0-51.46, P = 0.006). Likewise, the AA genotype was associated with advanced LF compared to the AG(OR = 3.60, 95%CI: 1.16-11.15, P = 0.02) or AG + GG genotypes(OR = 3.52, 95%CI: 1.18-10.45, P = 0.02).CONCLUSION This study suggests that the CD36(rs1761667) AA genotype is associated with higher fat intake and more instances of advanced LF in CHC patients.

Single amino acid mutation of SR-BI decreases infectivity of hepatitis C virus derived from cell culture in a cell culture model

AIM To investigate the effect of a single amino acid mutation in human class B scavenger receptor I(SR-BI) on the infectivity of cell culture-derived hepatitis C virus(HCVcc) in SR-BI knock-down Huh7-si SR-BI cells.METHODS Site-directed mutagenesis was used to construct the SR-BI S112 F mutation,and the mutation was confirmed by nucleotide sequencing. SR-BI knock-down Huh7-si SR-BI cells were transfected with SR-BI S112 F,SR-BI wild type(WT) and control plasmids,and then infected with HCVpp(HCV pseudoparticles) and hepatitis C virus derived from cell culture(HCVcc). A fluorescence assay was performed to analyze the effect of the S112 F mutation on HCV entry; quantitative real-time PCR,immunofluorescence,and Western blot assays were used to analyze the effect of the S112 F mutation on HCV infectivity. CHO cells expressing WT and SRBI S112 F were incubated with the HCV E2 protein expressed in HEK 293 T cells,and flow cytometry was performed to examine the ability of SR-BI S112 F to bind to the HCV E2 protein. Huh7-si SR-BI cells were transfected with SR-BI WT and the S112 F mutant,andthen Di I-HDL was added and images captured under the microscope to assess the ability of SR-BI S112 F to take up HDL.RESULTS The SR-BI S112 F mutation was successfully constructed. The S112 F mutation decreased the expression of the SR-BI m RNA and protein. SR-BI S112 F decreased HCV entry and HCVcc infectivity in Huh7-si SR-BI cells. The S112 F mutation impaired the binding of SR-BI to HCV E2 protein and decreased the HDL uptake of SR-BI.CONCLUSION The S112 F single amino acid mutation in SR-BI decreased the levels of the SR-BI m RNA and protein,as well as the ability of SR-BI to bind to the HCV E2 protein. Amino acid 112 in SR-BI plays important roles in HCV entry and the infectivity of HCVcc in vitro.

DC-SIGN: binding receptors for hepatitis C virus

Objective To review the recent developments in and research into binding receptors of hepatitis C virus (HCV) and especially the role of dendritic cell-specitic adhesion receptor (DC-SIGN) in HCV.Data sources Both Chinese- and English-languge literature was searched using MEDLINE (2000-2003) and the databank of Chinese-language literature (2000-2003).Study selection Relevant articles on DC-SIGN and HCV binding receptors in recent domestic and foreign literature were selected.Data extraction Data were mainly extracted from 40 articles which are listed in the references section of this review. Results DC-SIGN, a dendritic cell-specific adhesion receptor and a type Ⅱ transmembrane mannose-binding C-type lectin, is very important in the function of dendritic cells (DC), both in mediating nave T cell interactions through ICAM-3 and as a rolling receptor that mediates the DC-specific ICAM-2-dependent migration processes. It can be used by HCV and other viral and bacterial pathogens including human immunodeficiency virus (HIV), Ebola virus, CMV and Mycobacterium tuberculosis to facilitate infection. Both DC-SIGN and DC-SIGNR can act either in cis, by concentrating virus on target cells, or in trans, by transmission of bound virus to a target cell expressing appropriate entry receptors. Recent report showed that DC-SIGN not only plays a role in entry into DC, HCV E2 interaction with DC-SIGN might also be detrimental to the interaction of DC with T cells during antigen presentation. Conclusions DC-SIGNs are high-affinity binding receptors for HCV.The clinical strategies that target DC-SIGN may be successful in restricting HCV dissemination and pathogenesis as well as directing the migration of DCs to manipulate appropriate immune responses in autoimmunity and tumorigenic situations.

Estrogen, estrogen receptors, and hepatocellular carcinoma: Are we there yet?

A protective role of the sex steroid hormone estrogenin hepatocellular carcinoma(HCC) was suggested a few decades ago according to clinical data showing higher HCC morbidity and mortality among males. Several recent studies further confirmed the anti-cancer effects of estrogen in the liver. However, it remains to be identified how to exploit estrogen signalling within clinical settings for HCC treatment. There are several unresolved issues related to the estrogen pathway in liver cells. The main problems include the absence of a clear understanding of which estrogen receptor(ER) isoform is predominantly expressed in normal and malignant liver cells, the ER isoform expression difference between males and females, and which ER isoform should be targeted when designing HCC therapy. Some of those questions were recently addressed by Iyer and coauthors. The current editorial review critically analyses the study by Iyer et al(WJG, 2017) that investigated the expression of ER subtypes in liver samples collected from patients with a healthy liver, hepatitis C virus cirrhosis, and HCC. ER presence was evaluated in association with gender, intracellular localization, inflammation marker NF-kB, and proliferation-related effector cyclin D1. The study limitations and advantages are discussed in light of recent advances in the HCC and estrogen signalling areas.

Role of transferrin receptor in hepatitis C viral infection

Hepatitis C virus(HCV) is the main pathogen causing chronic hepatitis and primary liver cancer. Various viral proteins and host cell molecules are involved in the HCV cell entry, but the mechanism of infection has not been completely elucidated. The transferrin receptor can act as a receptor for many viruses during cell entry. The transferrin receptor is not only closely related to HCV-induced iron metabolism disorders but also mediates the fusion of HCV with the host cell membrane as a specific receptor for CD81-dependent viral adhesion.

M2BPGi for assessing liver fibrosis in patients with hepatitis C treated with direct-acting antivirals

BACKGROUND Assessing liver fibrosis is important for predicting the efficacy of direct-acting antivirals(DAAs)and patient prognosis.Non-invasive techniques to assess liver fibrosis are becoming important.Recently,serum Mac-2 binding protein glycosylation isomer(M2BPGi)was identified as a non-invasive marker of liver fibrosis.AIM To investigate the diagnostic accuracy of M2BPGi in assessing liver fibrosis in patients with chronic hepatitis C(CHC)treated with DAAs.METHODS From December 2017 to August 2018,80 treatment-naive adult patients with CHC who were eligible for DAAs therapy were consecutively enrolled in this observational cohort study.For 12 weeks,65 patients were treated with sofosbuvir/daclatasvir,and 15 patients were treated with sofosbuvir/daclatasvir and a weight-based dose of ribavirin at knowledge and technology association for hepatitis C management clinic,Cairo,Egypt.We measured serum M2BPGi levels,PAPAS index,fibrosis-4(FIB-4)score and liver stiffness measurements(LSM)at baseline and 12 weeks after the end of treatment.Serum M2BPGi levels were measured using enzyme-linked immunosorbent assay.RESULTS All patients achieved sustained virologic response(SVR12)(100%).Serum M2BPGi levels,LSM,FIB-4 score and PAPAS index decreased significantly at SVR12(P<0.05).Serum M2BPGi levels correlated positively with LSM at baseline and SVR12(P<0.001).At baseline,compared with the FIB-4 score and PAPAS index,M2BPGi was the best marker to distinguish patients with grade F4 fibrosis(AUC=0.801,P<0.001),patients with grade F2 from grade F0-1 fibrosis(AUC=0.713,P=0.012),patients with grade F3-4 from grade F0-2 fibrosis(AUC=0.730,P<0.001),and patients with grade F2-4 from grade F0-1 fibrosis(AUC=0.763,P<0.001).At SVR12,M2BPGi had the greatest AUCs for differentiating patients with grade F4 fibrosis(AUC=0.844,P<0.001),patients with grade F3 from grade F0-2 fibrosis(AUC=0.893,P=0.002),patients with grade F3-4 from grade F0-2 fibrosis(AUC=0.891,P<0.001),and patients with grade F2-4 from grade F0-1 fibrosis(AUC=0.750,P<0.001).CONCLUSION M2BPGi is a reliable marker for the non-invasive assessment and prediction of liver fibrosis regression in patients with CHC who achieved an SVR with DAAs therapy.

Spike protein homology between the SARS-associated virus and murine hepatitis virus implies existence of a putative receptor-binding region

Coronavirus has been determined to be the cause of the recent outbreak of severe acute respiratory syndrome (SARS). Human coronavirus 229E had been studied well and its receptor-binding domain was restricted to aa417—547 of S protein. However, this region has no homology with the newly separated SARS-associated virus (Hong Kong isolate CUHK-W1). Then we analyzed the phylogenesis of S1 subunit of the coronavirus spike protein (SARS-associated virus, Hong Kong isolate CUHK-W1). Interestingly, the highest homology between murine hepatitis virus (MHV) and SARS-associated coronavirus was found. And the important sites (aa62—65 and aa214—216) on the spike protein of MHV with receptor-binding capacity were highly conservative in comparison with the newly separated SARS-asso- ciated virus (the corresponding sites are aa51—54 and aa195—197). These results from bioinformatics analysis might help us to study the receptor-binding sites of SARS-associ- ated virus and the mechanism of the virus entry into the target cell, and design antiviral drugs and potent vaccines.

Specific interaction of hepatitis C virus glycoproteins with mannan binding lectin inhibits virus entry

Mannan-binding lectin(MBL)is a soluble innate immune protein that binds to glycosylated targets.MBL acts as an opsonin and activates complement,contributing to the destruction and clearance of infecting microorganisms.Hepatitis C virus(HCV)encodes two envelope glycoproteins E1 and E2,expressed as non-covalent E1/E2 heterodimers in the viral envelope.E1 and E2 are potential ligands for MBL.Here we describe an analysis of the interaction between HCV and MBL using recombinant soluble E2 ectodomain fragment,the full-length E1/E2 heterodimer,expressed in vitro,and assess the effect of this interaction on virus entry.A binding assay using antibody capture of full length E1/E2 heterodimers was used to demonstrate calcium dependent,saturating binding of MBL to HCV glycoproteins.Competition with various saccharides further confirmed that the interaction was via the lectin domain of MBL.MBL binds to E1/E2 representing a broad range of virus genotypes.MBL was shown to neutralize the entry into Huh-7 cells of HCV pseudoparticles(HCVpp)bearing E1/E2 from a wide range of genotypes.HCVpp were neutralized to varying degrees.MBL was also shown to neutralize an authentic cell culture infectious virus,strain JFH-1(HCVcc).Furthermore,binding of MBL to E1/E2 was able to activate the complement system via MBL-associated serine protease 2.In conclusion,MBL interacts directly with HCV glycoproteins,which are present on the surface of the virion,resulting in neutralization of HCV particles.

Distinct toll-like receptor expression in monocytes and T cells in chronic HCV infection

AIM： Hepatitis C virus often establishes chronic infections. Recent studies suggest that viral and bacterial infections are more common in HCV-infected patients compared to controls. Pathogens are recognized by Toll-like receptors （TLRs） to shape adaptive and innate immune responses. METHODS： In this study, to infected host to recognize assess the ability of HCV-infected host to recognize invading pathogens, we investigated Toll-like receptor expression in innate （monocytes） and adaptive （T cells） immune cells by realtime PCR. RESULTS： We determined that RNA levels for TLRs 2, 6. 7, 8, 9 and 10 mRNA levels were upregulated in both monocytes and T cells in HCV-infected patients compared to controls. TLR4 was only upregulated in T lymphocytes, while TLR5 was selectively increased in monocytes of HCV-infected patients. MD-2, a TLR4 coreceptor, was increased in patients＇ monocytes and T cells while CD14 and MyD88 were increased only in monocytes. CONCLUSION： Our data reveal novel details on TLR expression that likely relates to innate recognition of pathogens and immune defense in HCV-infected individuals.

Phage display selection on whole cells yields a small peptide specific for HCV receptor human CD81

The human CD81(hCD81),the most recently proposed receptor of hepatitis C virus(HCV),can especifically bind to HCV envelope glycoprotein 2(E2).In this study,hCD81-expressing murine NIH/3T3 cells were used to select hCD81-binding peptides from a phage displayed nonapeptide library(PVIII9aaCys).Eighteen of the 75clones selected from the library showed specific binding to the hCD81-expressing NIH/3T3 cells by enzyme linked immunosorbent assay(ELISA)and competitive inhibition test.Twelve out of the 18 clones shared the amino acid motif SPQYWTGPA.Sequence comparison of the motif showed no amino acid homology with the native HCV E2.The motif-containing phages could competitively inhibit the ability of HCV E2 binding to native hCD81-expressing MOLT-4 cells,and induce HCV E2 specific immune response in vivo.These results suggest that the selected motif SPQYWTGPA should be a mimotope of HCV E2 to bind to hCD81 molecules.Our findings cast new light on developing HCV receptor antagonists.