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 glycoprotein2 (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 75 clones 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.     

Selection of a phage-displayed peptide recognized by monoclonal antibody directed blocking the site of hepatitis C virus E2 for human CD81

The human CD81 (hCD81) molecule has been identified as a putative receptor for hepatitis C virus (HCV). HCV envelope glycoprotein 2 (E2) most likely plays a pivotal role in binding to host cells by interacting with the hCD81 molecule. In this study, a phage-displayed peptide library was used to select small peptides with anti-hCD81 monoclonal antibody JS-81. The output/input ratio of phages increased about 91 fold after the third round of selection. Eight of the 30 phage clones selected from the phage library showed specific binding to the anti-hCD81 by enzyme linked immunosorbent assay (ELISA). Competitive inhibition test further demonstrated that HCV E2 could significantly inhibit the binding of a positive phage clone to anti-hCD81 JS-81. Exogenous small peptide ATWVCGPCT contained by the positive phage clones showed aligned with the hCD81 sequence from 153-161 by sequence analyses. These results suggest that the selected ATWVCGPCT is a novel hCD81-like small peptide, which can block the binding site of HCV E2 for hCD81. It may be of further application on development of antiviral agents targeting the stage of HCV entry.     

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.     

Tupaia CD81, SR-BI, claudin-1, and occludin support hepatitis C virus infection

Hepatitis C virus (HCV)-related research has been hampered by the lack of appropriate small-animal models. It has been reported that tree shrews, or tupaias (Tupaia belangeri), can be infected with serum-derived HCV. However, these reports do not firmly establish the tupaia as a reliable model of HCV infection. Human CD81, scavenger receptor class B type I (SR-BI), claudin 1 (CLDN1), and occludin (OCLN) are considered essential receptors or coreceptors for HCV cell entry. In the present study, the roles of these tupaia orthologs in HCV infection were assessed. Both CD81 and SR-BI of tupaia were found to be able to bind with HCV envelope protein 2 (E2). In comparison with human CD81, tupaia CD81 exhibited stronger binding activity with E2 and increased HCV pseudoparticle (HCVpp) cell entry 2-fold. The 293T cells transfected with tupaia CLDN1 became susceptible to HCVpp infection. Moreover, simultaneous transfection of the four tupaia factors into mouse NIH 3T3 cells made the cells susceptible to HCVpp infection. HCVpp of diverse genotypes were able to infect primary tupaia hepatocytes (PTHs), and this infection could be blocked by either anti-CD81 or anti-SR-BI. PTHs could be infected by cell culture-produced HCV (HCVcc) and did produce infectious progeny virus in culture supernatant. These findings indicate that PTHs possess all of the essential factors required for HCV entry and support the complete HCV infection cycle. This highlights both the mechanisms of susceptibility of tupaia to HCV infection and the possibility of using tupaia as a promising small-animal model in HCV study.     

Involvement of somatically acquired ecotropic viruses in the immunogenicity of nude-transplanted NIH/3T3 transformed cell lines.

Immunogenic tumor variants were previously derived after transplantation in vivo into nude mice of NIH/3T3-transformed cell lines. Nude-passaged cell lines were rejected by immunocompetent H-2q NIH mice, were recognized by specific CTL clones, and expressed new retroviral Ag. The aim of the present work was to investigate whether somatically acquired proviral sequences were present in the genome of nude-passaged cells and to test directly for a causative relationship between murine leukemia virus (MuLV) expression and immunogenicity. Southern blot analysis of PstI-digested DNA indicated that in contrast to the parental NIH/3T3 transformed cell lines (pT, T12N/5a, NS-1) all the nude-passaged immunogenic variants (pT-nude, T12N/5a-nude, NS-1-nude) contained newly acquired ecotropic-related proviruses. Immediately after in vitro establishment, these tumors displayed multiple integration sites as assessed by analysis of 3' proviral-cellular junctions. Long term in vitro culture of one of the cell lines (pT-nude) resulted in a cell line (pT-nude/vitro) that was clonal or oligo-clonal with respect to viral integration. Northern blot analysis established that the new proviruses were actively transcribed in all the immunogenic variants. To assess whether the somatically acquired ecotropic proviral sequences encode for target structures recognized by specific CTL, obtained after immunization of NIH mice with pT-nude, the parental cell line pT was transfected with plasmids containing the entire AKV MuLV genome, the cloned AKV gag or env genes. Screening of transfectants for their ability to stimulate the production of TNF by anti-pT-nude effectors indicated that cells transfected with the entire ecotropic virus or with MuLV-env gene products could be recognized by an NIH anti-pT-nude CTL line and NIH anti-pT-nude Kq-restricted CTL clones as well as the immunizing target pT-nude.     

CD81 expression is important for the permissiveness of Huh7 cell clones for heterogeneous hepatitis C virus infection

Huh7 cells constitute a permissive cell line for cell culture of hepatitis C virus (HCV) particles. However, our Huh7 line shows limited permissiveness for HCV. Thus, in this study we set out to determine which host factors are important for conferring permissiveness. To analyze the limited permissiveness of our Huh7 cells, 70 clones were obtained after single-cell cloning of parental Huh7 cells. The cloned Huh7 cells exhibited various levels of HCV pseudoparticles and JFH-1 virus infection efficiency, and some clones were not permissive. A subgenomic replicon was then transfected into the cloned Huh7 cells. While the replication efficiencies differed among the cloned Huh7 cells, these efficiencies did not correlate with infectious permissibility. Flow cytometry showed that CD81, scavenger receptor class B type I, and low-density-lipoprotein receptor expression on the cell surfaces of the Huh7 clones differed among the clones. Interestingly, we found that all of the permissive cell clones expressed CD81 while the nonpermissive cell clones did not. To confirm the importance of CD81 expression for HCV permissiveness, CD81 was then transiently and stably expressed on a nonpermissive Huh7 cell clone, which was consequently restored to HCV infection permissiveness. Furthermore, permissiveness was down-regulated upon transfection of CD81 silencing RNA into a CD81-positive cell clone. In conclusion, CD81 expression is an important determinant of HCV permissiveness of Huh7 cell clones harboring different characteristics.     

稳定表达小鼠CD40L的NIH3T3细胞系的建立及其在B细胞培养和激活中的应用

该研究构建小鼠CD40L真核表达重组质粒pcDNA3.1-mCD40L,通过电转法将重组质粒转至NIH3T3细胞中。利用G418对转染后细胞进行压力筛选,获得稳定转染细胞株。提取稳定转染细胞株RNA,通过RT-PCR法检测Neo基因的mRNA表达情况。分离稳定转染细胞上清,利用ELISA法检测小鼠CD40L蛋白水平的表达情况。RT-PCR结果显示,Neo基因能够在稳定转染细胞中表达,ELISA结果显示,获得的稳定转染细胞株NIH3T3-mCD40L细胞上清中CD40L的表达量高达1.286 ng/mL。进一步活性研究表明,该细胞系能够在体外与IL-2和IL-21共同作用培养B细胞至14天,并刺激B细胞产生特异性抗体。该细胞系的成功构建,为利用体外B细胞分离培养和活化法分离特异性单克隆抗体奠定了良好的基础。     

EphA3基因稳定表达细胞模型的建立与分析

目的：建立稳定表达外源EphA3基因的小鼠成纤维细胞株模型,初步探讨EphA3基因表达对肿瘤发生、发展的影响。方法：通过脂质体介导的方法,将真核表达载体pcDNA3.1（-）/myc-his-EphA3转染NIH3T3细胞,用Western印迹确定外源EphA3基因表达;通过MTT实验、软琼脂集落形成实验,观察EphA3基因表达对NIH3T3细胞生物学特性的影响。结果：建立了稳定转染EphA3基因的NIH3T3细胞株;EphA3基因表达的小鼠成纤维NIH3T3细胞生长速度没有明显变化,但在软琼脂上锚着非依赖生长的能力加强。结论：建立了稳定表达外源EphA3基因的NIH3T3细胞株,EphA3基因稳定表达具有诱导正常NIH3T3细胞发生恶性转化的重要生物功能。     

Growth-Inhibitory Functions of a Mutated Gelsolin (His321) in NIH/3T3 Mouse Fibroblasts

We have previously established a murine flat revertant cell line R1 from an activated H-ras transformant EJ-NIH/3T3 by subjecting it to ethyl methanesulfonate. From the R1 cells, we cloned a mutated gelsolin gene His321 and have shown the inhibitory activity of His321 against EJ-NIH/3T3 tumors. Our present experiments were conducted to find out whether the His321 gene has any effects on untransformed NIH/3T3 fibroblasts. Rhodamine-phalloidin staining revealed that two NIH/3T3 clones expressing His321 (NIH/λ2S-3 and NIH/λ2S-6) form organized actin stress fibers as two clones transfected with the vector alone (NIH/neo-3 and NIH/neo-5). We also found that in a liquid medium, NIH/λ2S-3 and NIH/λ2S-6 grew more slowly than NIH/neo-3 and NIH/neo-5 and that the doubling times of the former were about 10 h slower than those of the latter. To investigate the effects of His321 on the signal transduction pathway necessary for cell growth, we stimulated the cell lines by prostaglandin E1 (PGE1), a platelet-derived growth factor (PDGF), or the epidermal growth factor (EGF). Although stimulation by PGE1 increased intercellular cyclic AMP in R1 cells, it did not do so in NIH/λ2S-3 and NIH/λ2S-6 cells. On the other hand, stimulation by PDGF or EGF induced far less DNA synthesis in NIH/λ2S-3 and NIH/λ2S-6 than in NIH/neo-3 and NIH/neo5. These results suggest that through the effects on the signal transduction pathway of PDGF and/or EGF His321-mutated gelsolin inhibits the growth of NIH/3T3.     

pcDNA3.1/myc-His-DJ-1^M26I重组载体构建及其对NIH3T3细胞增殖和凋亡研究

目的构建pcDNA3.1/myc-His-DJ-1和pcDNA3.1/myc-His-DJ-1M26I重组表达载体,为研究DJ-1M26I突变与细胞增殖、凋亡的关系及建立转基因动物模型奠定基础。方法采用突变试剂盒将DJ-1蛋白第26位氨基酸进行突变,分别构建pcDNA3.1/myc-His-DJ-1和pcDNA3.1/myc-His-DJ-1M26I重组表达载体,并采用脂质体介导的方法分别将其转染入NIH3T3细胞,500μg/mL G418压力筛选稳定克隆,对2种转染细胞在DNA水平、RNA水平和蛋白质水平进行鉴定,采用MTT染色方法和AnnexinV-FITC试剂盒进行转染阳性克隆细胞的细胞活力与细胞凋亡检测。结果 pcDNA3.1/myc-His-DJ-1和pcDNA3.1/myc-His-DJ-1M26I重组质粒转染NIH3T3细胞经G418筛选后,PCR方法检测分别获得1个和3个阳性细胞克隆,RT-PCR及western blot方法进行DJ-1-His基因表达检测,结果均证明外源插入基因的表达,MTT实验结果初步证明转染DJ-1M26I基因的NIH3T3阳性细胞组细胞增殖速率低于正常NIH3T3细胞组（P〈0.05）,转染DJ-1基因的NIH3T3阳性细胞组细胞增殖速率与正常NIH3T3细胞相比无明显差别;细胞凋亡检测表明转染DJ-1M26I基因的NIH3T3阳性细胞组细胞凋亡率高于正常NIH3T3细胞,转染DJ-1基因的NIH3T3阳性细胞组细胞凋亡率低于正常NIH3T3细胞（P〈0.05）。结论成功构建pcDNA3.1/myc-His-DJ-1和pcDNA3.1/myc-His-DJ-1 M26I重组表达载体,成功筛选出稳定表达人DJ-1及DJ-1 M26I的NIH3T3细胞。DJ-1 M26I基因突变更易导致NIH3T3细胞的凋亡。     

大鼠催乳素基因真核细胞可表达性质粒的构建及应用研究

７３５ｂｐ的ＰＲＬｃＤＮＡ片段从质粒ＰＲＬ－ＳＰ６５＃１中回收后,用粘性末端连接法将其重组到真核表达载体ｐｃＤＮＡ３上,筛选出正向连接重组体ｐｃＤＮＡ３－ＰＲＬＳ和反向连接重组体ｐｃＤＮＡ３－ＰＲＬＡＳ。将重组体ｐｃＤＮＡ３－ＰＲＬｓ和空载体ｐｃＤＮＡ３分别转入ＮＩＨ３Ｔ３细胞系,用Ｇ４１８筛选出阳性细胞后与未转染的ＮＩＨ３Ｔ３细胞在加Ｅ２和不加Ｅ２的情况下,用原位杂交的方法,分别用ＰＲＬｃＤＮＡ探针和原癌基因ｃ－Ｈ－ｒａｓｃＤＮＡ探针进行检测,未转染的ＮＩＨ３Ｔ３细胞在加Ｅ２和不加Ｅ２时都几乎无催乳素基因的表达,同样,转入空载体的ＮＩＨ３Ｔ３细胞也无ＰＲＬ的表达,而转入重组体ｐｃＤＮＡ３－ＰＲＬＳ的ＮＩＨ３Ｔ３细胞则有大量的ＰＲＬ基因的表达,与对照组相比有显著差异（Ｐ＜０．０１）。正常和转入空载体的ＮＩＨ３Ｔ３细胞有一定程度的原癌基因ｃ－Ｈ－ｒａｓ的表达,当分别加入Ｅ２和转入重组体ｐｃＤＮＡ３－ＰＲＬＳ后,ＮＩＨ３Ｔ３细胞中的ｃ－Ｈ－ｒａｓ基因表达水平都显著升高（Ｐ＜０．０５）。     

人sTNFR1基因的克隆以及在NIH3T3细胞中的稳定表达

以He1a细胞的总RNA为模板,用RT—PCR方法扩增sTNFR1全编码区基因片段,构建含有目的片段的T载体克隆及真核表达载体pcDNA3．1（-）重组质粒亚克隆,将重组质粒和脂质体共同转染NIH3T3细胞系,G418筛选稳定转染细胞株．经核苷酸序列测序和酶切鉴定,成功构建了pcDNA3．1（-）-sTNFR1真核表达质粒,脂质体法建立了高效表达sTNFRI的稳定转染细胞系,并经RT—PCR和Western Blotting鉴定．人sTNFR1基因能在NIH3T3细胞系中稳定表达,为今后的研究打下了基础．     

Expression of retrovirus-related, cytotoxic T lymphocyte- and transplantation-defined antigens in NIH/3T3 transfectants after a single passage in nude mice

Transfection of T24c-Ha-ras oncogene into NIH/3T3 fibroblasts resulted in the establishment of a transformed cell line (pT) that was tumorigenic when injected s.c. both into Swiss outbred nude mice and normal NIH inbred mice. The passage into nude mice, however, led to the development of a tumor variant (pT-nude) able to subsequently grow into sublethally x-irradiated but not into immunocompetent NIH mice. NIH mice immunized with this tumor variant developed a strong specific CTL response against the immunizing cell line, whereas the parental transformed pT cell line was not lysed. Clones were derived by limiting dilution from anti-pT-nude bulk population and were tested on a panel of transformed NIH/3T3 lines before and after their growth as tumor into nude mice. All of these lines were lysed by the Lyt-2+ CTL clones as a sole consequence of one in vivo passage into nude mice. The cross-reactive Ag were shown to be related to endogenous retroviral products as assessed by 1) immunoprecipitations of gp70, p15E, and p30 viral proteins in the nude variants but not in parental lines, and 2) by the ability of retroviruses from irradiated pT-nude cells to infect NIH/3T3 or pT lines making them susceptible to lysis by anti-pT-nude CTL clones. These results show that a single passage in nude mice can induce retrovirus-related, cell-surface Ag in transplanted neoplastic cells.     

DJ-1~（L166P）与DJ-1~（M26I）基因对NIH3T3细胞增殖和凋亡的比较

目的在细胞学水平比较DJ、DJ-1M26 I、DJ-1L166 P基因对NIH 3T3细胞增殖速率与凋亡的关系,为建立转基因动物模型及帕金森疾病发病机制研究奠定基础。方法分别将pcDNA3.1/myc-His-DJ-1、pcDNA3.1/myc-His-DJ-1L166 P和pcDNA3.1/myc-His-DJ-1M26 I重组质粒脂质体方法转染NIH 3T3细胞,500μg/ml G418压力筛选稳定克隆,对3种转染细胞在DNA水平、RNA水平和蛋白质水平进行鉴定,采用MTT染色方法和Annexin V-FITC试剂盒进行转染阳性克隆细胞的细胞活力与细胞凋亡检测。结果 pcDNA3.1/myc-His-DJ-1、pcDNA3.1/myc-His-DJ-1L166 P和pcDNA3.1/myc-His-DJ-1M26 I重组质粒转染NIH 3T3细胞经G418筛选后,PCR方法检测分别获得1个、4个、3个阳性细胞克隆,RT-PCR及Western blot方法进行DJ-1-His基因表达检测,结果均证明外源插入基因的表达,Caspase-3 RNA水平检测DJ-1L166 P和DJ-1M26 I组表达高于正常NIH 3T3细胞组,而DJ-1组caspase-3转录水平相对最低,MTT实验结果初步证明转染DJ-1L166 P和DJ-1M26 I基因的NIH3T3阳性细胞组细胞增殖速率均低于DJ-1组和正常NIH 3T3细胞组（P〈0.05）,转染DJ-1基因的NIH 3T3阳性细胞增殖速率与正常NIH 3T3细胞相比无明显差别;细胞凋亡检测表明转染DJ-1L166 P和DJ-1M26 I基因的NIH3T3阳性细胞凋亡率均高于正常NIH 3T3细胞,转染DJ-1基因的NIH 3T3阳性细胞凋亡率低于正常NIH 3T3细胞（P〈0.05）。结论 DJ-1L166 P和DJ-1M26 I基因突变均降低NIH3T3细胞增殖速率,DJ-1L166 P和DJ-1M26 I基因突变更易导致NIH 3T3细胞的凋亡,DJ-1L166 P和DJ-1M26 I基因突变对NIH3T3细胞增殖速率和细胞凋亡影响是相似的。     

ERK signaling is triggered by hepatitis C virus E2 protein through DC-SIGN

Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is a binding receptor for hepatitis C virus (HCV). Binding of HCV envelope protein E2 to target cells is a prerequisite to DC-SIGN-mediated signaling. Using cell lines with stable or transient expression of DC-SIGN, we investigated effects of soluble HCV E2 protein on ERK pathway. MEK and ERK are activated by the E2 in NIH3T3 cells stably expressing DC-SIGN. Treatment of the cells with antibody to DC-SIGN results in inhibition of the E2 binding as well as the E2-induced MEK and ERK activation. In HEK293T cells transiently expressing DC-SIGN, activation of MEK and ERK is also induced by the E2. Activation of ERK pathway by HCV E2 through DC-SIGN provides useful information for understanding cellular receptor-mediated signaling.     

Correlation of the tight junction-like distribution of Claudin-1 to the cellular tropism of hepatitis C virus

Claudin-1 (CLDN1), a tight junction (TJ) protein, has recently been identified as an entry co-receptor for hepatitis C virus (HCV). Ectopic expression of CLDN1 rendered several non-hepatic cell lines permissive to HCV infection. However, little is known about the mechanism by which CLDN1 mediates HCV entry. It is believed that an additional entry receptor(s) is required because ectopic expression of CLDN1 in both HeLa and NIH3T3 cells failed to confer susceptibility to viral infection. Here we found that CLDN1 was co-immunoprecipitated with both HCV envelope proteins when expressed in 293T cells. Results from biomolecular fluorescence complementation assay showed that overexpressed CLDN1 also formed complexes with CD81 and low density lipoprotein receptor. Subsequent imaging analysis revealed that CLDN1 was highly enriched at sites of cell-cell contact in permissive cell lines, co-localizing with the TJ marker, ZO-1. However, in both HeLa and NIH3T3 cells the ectopically expressed CLDN1 appeared to reside predominantly in intracellular vesicles. The CLDN1-CD81 complex formed in HeLa cells was also exclusively distributed intracellularly, co-localizing with EEA1, an early endosomal marker. Correspondingly, transepithelial electric resistance, obtained from the naturally susceptible human liver cell line, Huh7, was much higher than that of the HeLa-CLDN1 cell line, suggesting that Huh7 is likely to form functional tight junctions. Finally, the disruption of TJ-enriched CLDN1 by tumor necrosis factor-alpha treatment markedly reduced the susceptibility of Huh7.5.1 cells to HCV infection. Our results suggest that the specific localization pattern of CLDN1 may be crucial in the regulation of HCV cellular tropism.     

Tet调控CYP2E1基因表达细胞系的代谢能力分析

应用Tet-On基因表达系统,调控CYP2E1基因在NIH 3T3细胞中的表达水平,探讨CYP2E1在化学致癌物二甲基亚硝胺(N-nitrosodimethylamine, NDMA)代谢中的作用．先后将 Tet-on基因表达系统的调控质粒pRevTet-on和反应质粒pRevTRE-2E1转染NIH 3T3细胞,分别用G418和潮霉素筛选,并通过RT-PCR和Western印迹检测,成功获得了3个具有良好诱导性Tet调控的CYP2E1基因表达细胞系（Tet/3T3-2E1）．应用不同浓度强力霉素(doxycycline, Dox)处理Tet/3T3-2E1细胞诱导CYP2E1表达,HPLC分析细胞对CYP2E1特异性药物探针氯唑沙腙的原位代谢能力及MTT法分析CYP2E1介导的NDMA细胞毒性作用．结果显示,Tet/3T3-2E1细胞CYP2E1的表达及其代谢能力有明显的Dox浓度依赖性,而且NDMA对Dox诱导组细胞有明显浓度依赖性细胞毒性作用,其IC₅₀值为12.06 μmol/L,无Dox诱导组未见明显的NDMA细胞毒性作用．该细胞模型的成功建立对于开展与CYP2E1相关的毒物、致癌物代谢研究,筛选抗毒物、抗癌物等具有重要价值．     

Interaction of L-SIGN with Hepatitis C Virus Envelope Protein E2 Up-Regulates Raf–MEK–ERK Pathway

Liver/lymph node-specific intercellular adhesion molecule-3-grabbing integrin (L-SIGN) facilitates hepatitis C virus (HCV) infection through interaction with HCV envelope protein E2. Signaling events triggered by the E2 via L-SIGN are poorly understood. Here, kinase cascades of Raf–MEK–ERK pathway were defined upon the E2 treatment in NIH3T3 cells with stable expression of L-SIGN. The E2 bound to the cells through interaction with L-SIGN and such binding subsequently resulted in phosphorylation and activation of Raf, MEK, and ERK. Blockage of L-SIGN with antibody against L-SIGN reduced the E2-induced phosphorylation of Raf, MEK, and ERK. In the cells infected with cell culture-derived HCV, phosphorylation of these kinases was enhanced by the E2. Up-regulation of Raf–MEK–ERK pathway by HCV E2 via L-SIGN provides new insights into signaling cascade of L-SIGN, and might be a potential target for control and prevention of HCV infection.     

Hepatitis C virus nonstructural protein NS3 transforms NIH 3T3 cells.

Clinical evidence suggests that hepatitis C virus (HCV) is etiologically involved in hepatic cancer and liver cirrhosis. To investigate whether the HCV nonstructural protein NS3 has oncogenic activity, NIH 3T3 cells were transfected with an expression vector containing cDNA for the 5'- or 3'-half sequence of the HCV genome segment encoding NS3. Only cells transfected with the 5'-half cDNA rapidly proliferated, lost contact inhibition, grew anchorage independently in soft agar, and formed tumors in nude mice. PCR analysis confirmed the presence of the 5'-half DNA in the transfectants. These results suggest that the 5' region of the HCV genome segment encoding NS3 is involved in cell transformation.     

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.