Hepatitis C virus core protein fused to hepatitis B virus core antigen for serological diagnosis of both hepatitis C and hepatitis B infections by ELISA

The sequence encoding the truncated core protein (amino acids 1–98) of hepatitis C virus (HCc) was expressed in E. coli for production of HCc_(1–98), or fused with the truncated core antigen (HBcAg) and segments from the preS1 and preS2 regions from hepatitis B virus (HBV) for production of HBcPreS₁PreS₂HCc_(1–98). The HCc_(1–98) and HBcPreS₁PreS₂HCc_(1–98) proteins reacted with sera from HCV-infected individuals by immunoblot analyses, while the latter protein also exhibited HBV core antigenicity. They induced antibodies against HBcAg and/or HCV core protein in rabbits and in mice. Moreover, HBcPreS₁PreS₂HCc_(1–98) is more immunogenic than HCc_(1–98) in terms of anti-HCc induction. An ELISA that employed recombinant HCV core antigens of either HCc_(1–98) or HBcPreS₁PreS₂HCc_(1–98) to detect anti-HCc and/or anti-HBc antibodies was developed. Evaluation of serum samples with different status of HBV and HCV infections suggested that HCc_(1–98) might be suitable for the determination of antibodies against HCV core protein, while HBcPreS₁PreS₂HCc_(1–98) might be of value to detect HCV and/or HBV infection in donated blood in HBV low-prevalence countries. J. Med. Virol. 57:104–110, 1999. © 1999 Wiley-Liss, Inc.     

The effect of mouse interferon on the transformation of NIH/3T3 mouse cells by murine sarcoma virus.

The effect of interferon (IF) on transformation of NIH/3T3 cells by murine sarcoma virus (MSV) was studied. Two assays of MSV transformation independent of virus replication were used, focus formation under conditions where foci developed from cells infected with MSV alone and colony formation in agar suspension medium. Pretreatment of cells with purified IF (10⁴ units/ml) substantially inhibited transformation upon infection with MSV by about 90%. Reconstruction experiments showed that such IF treatment did not block focus or colony formation by cells already stably transformed by MSV. These results indicate that there is an IF sensitive early step in the transformation process.     

Phosphorylation of hepatitis C virus core protein by protein kinase A and protein kinase C

Hepatitis C virus (HCV) core protein can form capsid-like particles and is believed to be the viral capsid protein. Besides its structural functions, this protein is also known to possess multiple regulatory functions. In this article, we have studied the possible phosphorylation of HCV core protein in two different human liver-derived cell lines Huh7 and HepG2. Our results indicated that the HCV core protein could be phosphorylated, albeit inefficiently, independent of its downstream E1 protein in these two cell lines. Two of the basal phosphorylation sites were identified to be serine-53 and serine-116. The phosphorylation of the core protein could be enhanced by the PKC activator phorbol 12-myristic 13-acetate (PMA), and the PKA activator forskolin, and these enhancements could be abolished by the respective inhibitors of PKC and PKA, indicating that the core protein is a substrate of these two kinases. While both serine-53 and serine-116 served as the PKC phosphorylation sites, serine-116 appeared to be the major PKA phosphorylation site. Further analyses using serine-to-alanine mutation to mimic dephosphorylation and serine-to-aspartic acid mutation to mimic phosphorylation revealed that the conversion of serine-116 to aspartic acid led to an enhanced nuclear localization of the core protein. This observation indicates that one function of phosphorylation may be to regulate the nuclear localization of the core protein.     

Increased liver pathology in hepatitis C virus transgenic mice expressing the hepatitis B virus X protein

Transgenic mice expressing the full-length HCV coding sequence were crossed with mice that express the HBV X gene-encoded regulatory protein HBx (ATX mice) to test the hypothesis that HBx expression accelerates HCV-induced liver pathogenesis. At 16 months (mo) of age, hepatocellular carcinoma was identified in 21% of HCV/ATX mice, but in none of the single transgenic animals. Analysis of 8-mo animals revealed that, relative to HCV/WT mice, HCV/ATX mice had more severe steatosis, greater liver-to-body weight ratios, and a significant increase in the percentage of hepatocytes staining for proliferating cell nuclear antigen. Furthermore, primary hepatocytes from HCV, ATX, and HCV/ATX transgenic mice were more resistant to fas-mediated apoptosis than hepatocytes from nontransgenic littermates. These results indicate that HBx expression contributes to increased liver pathogenesis in HCV transgenic mice by a mechanism that involves an imbalance in hepatocyte death and regeneration within the context of severe steatosis.     

HCV NS3 N端多肽诱导人肝细胞系转化及成瘤实验

目的 研究丙型肝炎病毒(HCV)非结构区3N端多肽(HCV NS3-5′)对人肝细胞株QSG7701的转化作用及致癌性。方法 通过脂质体介导将含有HCV NS3 N端cDNA的真核表达质粒(pRcHCNS3-5′)导入人源性肝细胞株QSG7701,G418筛选目的基因表达的细胞;聚合酶链反应(PCR)及免疫组织化学SP法检测细胞中HCV NS3基因及蛋白的表达;细胞计数,锚着非依赖性生长实验,成瘤性检测等鉴定其生物学行为变化,免疫组织化学S-P法检测所致肿瘤中HCV NS3及c-myc蛋白表达。结果 HCV NS3-5′转染的QSG7701细胞中NS3蛋白过度表达于胞质,质粒pRcHCNS3-5′转染细胞的倍增时间较pRcCMV转染细胞和未转染QSG7701细胞明显缩短(分别为12h,26h,28h)。pRcHCNS3-5′和pRcCMV转染细胞及未转染QSG7701在软琼脂中的克隆形成率分别为33.0％、1.5％、1.1％。pRcHCNS3-5′转染细胞的克隆率高于其他两种转染细胞(P<0.01)。三种细胞接种探鼠后,pRcHCNS3-5′转染细胞注射组出现肿瘤,为肝细胞癌,肿瘤组织有HCV NS3蛋白和c-myc蛋白的表达。阳性对照组亦出现肿瘤,而pRcCMV转染细胞及未转染QSG7701细胞注射组在注射40d后仍未见肿瘤发生。结论 HCV NS3 N端蛋白具有转化细胞和促进肿瘤发生的作用。     

HCV核心蛋白与NS4B对HepG2细胞增殖的影响

目的 探讨HCV核心蛋白与非结构蛋白4B（NS4B）对HepG2细胞增殖的影响及可能机制.方法 重组质粒pcDNA3.1（-）Core与pcDNA3.1（-）NS4B单独和联合转染HepG2细胞,同时以转染空载体和未转染HepG2细胞作为对照.RT-PCR及Western Blot检测各组细胞中HCVCore、NS4B 、Wnt1、β-catenin 、c-myc及CyclinD1表达;MTT法,平板克隆形成试验检测HCV核心蛋白与NS4B对HepG2细胞增殖的影响;流式细胞术检测细胞周期.结果 ①pcDNA3.1（-）Core与pcDNA3.1（-）NS4B单独和联合转染HepG2细胞,成功表达HCV Core或/（和）NS4B mRNA和蛋白.②pcDNA3.1（-）Core和pcDNA3.1（-）NS4B单独转染和联合转染的HepG2细胞Wnt1、β-catenin、c-myc、CyclinD1 mRNA与蛋白的相对表达量均高于HepG2/pcDNA3.1（-）组和HepG2组（P＜0.01）.③与HepG2/pcDNA3.1（-）组和HepG2组比较,pcDNA3.1（-）Core和pcDNA3.1（-） NS4B单独转染和联合转染的HepG2细胞活力和克隆形成能力增强,S期和G2/M期细胞比例升高（P＜0.01）.结论 HCV核心蛋白与NS4B能加速HepG2细胞周期进程,促进细胞增殖,这种效应可能与其增强Wnt1、β-catenin、c-myc及CyclinD1的表达相关.     

Internalisation of hepatitis C virus core protein by human conjunctival fibroblasts

Recent studies indicate that hepatitis C virus (HCV) proteins can mediate innate immune response and inflammation in conjunctival fibroblasts which contributes to the pathology of dry eye condition associated with chronic HCV infection. The present study investigates the phagocytic potential of human conjunctival fibroblasts (HCFj) for HCV core protein. HCFj cells were incubated with HCV core antigen for different periods of time, and fluorescent micrographs were taken to observe protein internalisation. HCFj cells were capable of internalising HCV core antigen within 1 h; this gives an insight into another molecular mechanism which may contribute towards HCV-associated conjunctival inflammation.     

调节性T细胞在3型鼠肝炎病毒诱导的小鼠暴发型肝炎模型中作用的初步探讨

目的 检测调节性T细胞(Tr)在3型鼠肝炎病毒(MHV-3)诱导的小鼠暴发型肝炎模型中的比例变化及细胞因子表达,初步探讨Tr在该疾病模型中的作用.方法 通过腹腔注射MHV-3感染BALB/cJ小鼠诱导暴发型肝炎,观察小鼠的生存时间,检测血清谷丙转氨酶(ALT)、谷草转氨酶(AST)水平,利用苏木精-伊红(HE)染色法检测肝脏病理学改变,分离感染不同时间点外周血、脾脏以及肝脏中的淋巴细胞,利用流式细胞术来检测Tr的比例以及细胞因子IL-10表达水平.结果 BALB/cJ小鼠感染MHV-3后,全部在3～6d内死亡,血清ALT、AST水平随着感染时间延长逐渐升高,HE染色显示肝脏组织炎症及坏死程度逐渐加重,流式细胞术检测发现随着感染时间延长,小鼠肝脏中的Tr的比例明显升高.同时肝脏Tr分泌细胞因子1L-10的比例以及肝脏组织IL-10的mRNA水平逐渐升高.结论 MHV-3诱导的小鼠暴发型肝炎模型中Tr在肝脏中的比例和功能显著升高,这种代偿性升高提示Tr可能发挥调节机体过度免疫反应的功能.     

Cathepsin L is required for ecotropic murine leukemia virus infection in NIH3T3 cells

Recently it has been reported that a cathepsin B inhibitor, CA-074Me, attenuates ecotropic murine leukemia virus (Eco-MLV) infection in NIH3T3 cells, suggesting that cathepsin B is required for the Eco-MLV infection. However, cathepsin B activity was negative or extremely low in NIH3T3 cells. How did CA-074Me attenuate the Eco-MLV infection? The CA-074Me treatment of NIH3T3 cells inhibited cathepsin L activity, and a cathepsin L specific inhibitor, CLIK148, attenuated the Eco-MLV vector infection. These results indicate that the suppression of cathepsin L activity by CA-074Me induces the inhibition of Eco-MLV infection, suggesting that cathepsin L is required for the Eco-MLV infection in NIH3T3 cells. The CA-074Me treatment inhibited the Eco-MLV infection in human cells expressing the exogenous mouse ecotropic receptor and endogenous cathepsins B and L, but the CLIK148 treatment did not, showing that only the cathepsin L suppression by CLIK148 is not enough to prevent the Eco-MLV infection in cells expressing both of cathepsins B and L, and CA-074Me inhibits the Eco-MLV infection by suppressing both of cathepsins B and L. These results suggest that either cathepsin B or L is sufficient for the Eco-MLV infection.     

A functional hepatitis B virus X protein produced in insect cells.

We have constructed a recombinant baculovirus from Autographa californica nuclear polyhedrosis virus, called AcX, that expresses the gene encoding the hepatitis B virus X protein in infected Spodoptera frugiperda (Sf21AE) insect cells. A 16.5-kDa monomer and a 33-kDa dimer of the X protein were detected in extracts from AcX-infected cells on immunoblots using a polyclonal anti-X antibody. The biological activity of the insect cell-produced X protein was assayed by fusing AcX-infected Sf21AE cells with African green monkey kidney (CV-1) cells and then transfecting the fused cells with the reporter plasmid pSV2cat. The expression of the cat gene in CV-1:Sf21AE(AcX) fusions was seven times higher than that derived from CV-1:Sf21AE(AcMNPV) fusions, indicating that the insect cell-produced X protein was functional. The transactivation function of the insect cell-produced X protein was also verified by scrape-loading nuclear extracts of AcX-infected Sf21AE cells into pSV2cat-transfected CV-1 cells. Treatment of the AcX-infected cell nuclear extracts with anti-X antisera prior to scrape-loading eliminated the transactivating activity of the extracts. We conclude that the insect cell-produced X protein was functionally identical to that generated in mammalian cells.     

T cell dysfunction by hepatitis C virus core protein involves PD-1/PDL-1 signaling

Reports have shown that a negative T cell costimulatory pathway mediated by PD-1 (programmed death-1) and PDL-1 (programmed death ligand-1) is associated with T cell exhaustion and persistent viral infection. Persistent hepatitis C virus (HCV) infection in humans is also characterized by impaired T lymphocyte function, but the role of the PD-1 and PDL-1 pathway in HCV infection is unknown. Here we report that T cells isolated from chronically HCV-infected patients express significantly higher levels of PD-1 when compared with healthy donors. In addition, PD-1 and PDL-1 expression is upregulated on healthy donor T cells exposed to HCV core, a nucleocapsid protein that is immunosuppressive; upregulation of PD-1 is mediated through interaction of HCV core with the complement receptor, gC1qR. Importantly, T cell functions that are dysregulated by HCV core, including T cell activation, proliferation, and apoptosis, can be restored by blocking PD-1 and PDL-1 engagement. Our results indicate that HCV core can upregulate a key negative T cell signaling pathway associated with viral persistence and highly expressed on the T cells of persistently infected individuals. This upregulation of the PD-1 and PDL-1 pathway in humans represents a novel and perhaps common mechanism by which a virus usurps host machinery to facilitate persistence.     

Detection of mutations in hepatitis B virus enhancer 2/core promoter and X protein regions in patients with fatal hepatitis B virus infection

The enhancer 2/core promoter and the X protein regions located upstream of the precore and core regions in hepatitis B virus regulate expression of core/e antigen peptides. Mutations in the precore and core regions have been reported to be associated closely with the severity of type B hepatitis, and regions regulating expression of these peptides may also be involved in severe liver damage. Mutations were examined in regions that may be related to fatal liver diseases. Nucleotide sequences and deduced amino acid sequences from 20 patients with fatal type B hepatitis (12 with fulminant hepatitis and 8 with severe exacerbation) and 10 patients with self-limited acute hepatitis were analyzed. There were 50 nucleotide alterations in the enhancer 2/core promoter region of virus from 12 patients with fulminant hepatitis (average 4.1/case), 37 alterations in 8 patients with severe exacerbation (4.6/case), and 10 mutations in 10 cases of acute hepatitis (1.0/case). The numbers of amino acid mutations in X protein were 53 in 12 cases of fulminant hepatitis (4.4/case), 27 in 8 cases of severe exacerbation (3.3/case), and 9 in 10 cases of acute hepatitis (0.9/case). In fatal cases, approximately 50% of the nucleotide mutations were located within the region spanning nucleotides 1741-1777 (14.2% of the enhancer 2/core promoter region) and 30% of the amino acid mutations in X protein were located within the region containing codons 122-132 (7.1% of X protein). In addition to mutations in the precore and core regions, mutations in the enhancer 2/core promoter and the X protein regions may be associated with the pathogenesis of fatal B hepatitis infection.     

Rapid syncytium formation induced by Moloney murine sarcoma virus in 3T3/NIH cells and its delay by mouse interferon

Summary Moloney murine sarcoma virus (MSV) clone 124 was found to induce rapid syncytium formation upon infection of 3T3/NIH at a high multiplicity of infection. This effect became apparent, by light microscopy, within 1 to 2 hours, whereas by scanning electron microscopy, clusters of 4 to 25 cells were seen in their initial steps of syncytium formation within 20 to 30 minutes after virus addition. It appeared that the cell fusion was initiated by connection between the cells through virus bound to their surface. After a few more hours several neighbouring syncytia fused into giant cells containing over a hundred nuclei. Though MSV (124) stocks contained also some MLV it appeared that the syncytium inducing activity was related to the MSV particles. MLV particles were not only incapable to induce syncytium formation, they even interfered with this activity of MSV. The MSV-induced cell fusion required the adsorption of intact virions, but was independent on protein synthesis. Mouse interferon remarkably reduced the rate of syncytium development.With 3 Figures     

乙型肝炎病毒X蛋白抑制SFRP5启动子的活性

 目的:探讨乙型肝炎病毒X蛋白(HBx)抑制分泌型卷曲相关蛋白5(SFRP5)启动子活性的分子机制。方法:扩增不同长度的SFRP5启动子区片段,构建到萤光素酶报告基因载体pGL3-Basic上,并将构建好的重组质粒转染入正常肝细胞系LO2细胞,再分别感染编码HBx的腺病毒（Ad-HBx）或编码绿色荧光蛋白的腺病毒（Ad-GFP）,荧光显微镜下观察病毒感染效率,检测萤光素酶活性以观察HBx对SFRP5启动子活性的影响。结果:(1) 含不同长度的SFRP5启动子区截短突变报告质粒构建成功。(2) 截短突变报告质粒的萤光素酶活性与pGL3-Basic对照组相比,分别为(6.32±0.04)倍(-478 bp~+47 bp)、(5.79±0.32)倍(-811 bp~+47 bp)、(3.59±0.34)倍(-1 235 bp~+47 bp)、(3.86±0.39)倍(-1 677 bp~+47 bp)、(3.26±0.42)倍(-2 072 bp~+47 bp) (均P<0.05)。过表达HBx可以明显抑制SFRP5启动子活性,抑制率分别为44%(-478 bp~+47 bp) (P<0.05)、 46%(-811 bp~+47 bp) (P<0.05)、 28%(-1 235 bp~+47 bp)、 24%(-1 677 bp~+47 bp)和40%(-2 072 bp~+47 bp)。结论: HBx可以明显抑制SFRP5启动子区活性,可能导致SFRP5基因表达下调。     

丙型肝炎病毒核心蛋白在患者外周血单个核细胞内的核表达检测

目的　检测和研究丙型肝炎病毒 (hepatitisCvirus,HCV)核心蛋白在患者外周血单个核细胞 (peripheralbloodmononuclearcells ,PBMC)内核表达的意义 ,并探讨其与临床的关系。方法　对 6 6例慢性丙型肝炎患者PBMC标本进行免疫组化检测 ,并将HCV蛋白抗原定位分布情况与患者临床状况进行比较分析 ,对其中 2 7例患者PBMC进行HCVRNA和HCVAg的平行检测和分析。结果　免疫组化结果显示 ,慢性丙型肝炎患者PBMCHCVAg(core +NS3)阳性检出率为 77 2 7% (5 1 6 6 )。结果还证实 ,HCV核心蛋白均定位于胞核内 ,且呈强表达 ;NS3蛋白主要定位于胞质内 ,呈弱表达。当进行HCVAg在PBMC内定位情况与患者临床状况比较分析时显示 ,病情较重患者PBMC内核心蛋白表达阳性率 (35 2 9% )明显高于病情较轻者 (5 88% ) (P <0 0 0 1)。结论　HCV核心蛋白在PBMC内核表达与患者临床状况相关 ,提示其可能是丙型肝炎慢性化的一个指标 ,并可能在肝硬化和肝癌发生上起一定作用     

丙型肝炎病毒核心蛋白对HepG2细胞生长周期的影响

目的: 构建丙型肝炎病毒核心蛋白(HCV-core-1b)真核重组质粒,获得稳定表达HCV-core-1b的HepG2细胞株,观察HCV-core-1b对HepG2细胞株生长周期及cyclin D1 和pRb/p130表达的影响,探讨丙型肝炎病毒慢性感染的可能机制。方法: 将HCV-core-1b亚克隆入pBabe-Flag-puro载体,获得重组质粒pBabe-Flag-HCV-core-1b;将重组质粒转染病毒包装细胞Pheonix 293T,筛选获得分泌HCV-core-1b的病毒包装细胞株。利用包装细胞产生的病毒上清感染靶细胞,筛选后获得稳定表达HCV-core-1b的HepG2细胞株,流式细胞仪检测靶细胞生长周期的变化,Western blotting检测cyclin D1 和pRb/p130蛋白的表达。结果: 基因测序确认HCV-core-1b亚型基因编码区完整无移位,与标签蛋白Flag形成融合蛋白。HepG2-HCV-core细胞株成功表达Flag-HCV-core-1b蛋白,并导致细胞cyclin D1 和 pRb/p130的水平下调,显著改变了HepG2细胞生长周期,使细胞阻滞在G₀/G₁期。结论: 成功构建了pBabe-Flag-HCV-core-1b真核表达质粒,获得稳定表达Flag-HCV-core-1b融合蛋白的HepG2细胞。由于HCV-core-1b蛋白的表达,下调了HepG2细胞 cyclin D1和pRb/p130的表达,显著抑制HepG2细胞生长周期。     

Knock-down of hepatitis B virus X protein reduces the tumorigenicity of hepatocellular carcinoma cells

Hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC) in Southeast Asia and Hong Kong. Among the four proteins that are encoded by the HBV genome, HBV X (HBx) is the most potentially oncogenic factor. It is known that HBx plays an important role in hepatocarcinogenesis, but the exact functions and molecular mechanisms of HBx in HCC are not well understood. In this study, we constructed expression vectors for small hairpin RNAs (shRNA) against HBx and investigated their regulatory effects in PLC/PRF/5 HCC cells, which constitutively produce HBx. Our data show that this tool of RNA interference (RNAi) could successfully reduce the HBx mRNA and protein levels by 50-95%. RNAi targeting HBx in PLC/PRF/5 cells demonstrated significant reduction in cell proliferation, cell growth, anchorage-independent growth in soft agar, and tumour development in nude mice. In addition, depletion of HBx expression increased cell sensitivity to TNFalpha-mediated and serum-free-induced apoptosis, and reduced the expression levels of C-myc and Bcl-X(L). These findings suggest that HBx plays an important role in tumorigenicity and anti-apoptotic mechanisms in HCC.