丙型肝炎病毒功能基因在CHO细胞中的表达研究

丙型肝炎病毒（HCV）与宿主细胞因子的相互作用已经成为国内外研究的热点和难点。近期研究已经证实HCV的感染对宿主多种途径中基因的转录均能产生影响。为了进一步研究究竟是HCV中的哪些功能基因在与特定细胞因子的相互作用中起主导作用,构建了分别含有HCV Core、E1、E2、p7、NS2、NS3、NS4A、NS4B、NS5A和NS5B基因的真核表达质粒,分别转入宿主细胞CHO-K1中,在G418的选择压力下筛选获得稳定表达HCV单个蛋白的细胞系（10株）。PCR和RT-PCR可分别从稳定细胞系中检测到相应的HCV基因的DNA和mRNA,冻存和复苏不会造成HCV基因的丢失。Western-blot检测到稳定细胞系中表达E1,E2和NS5B蛋白,说明HCV基因在CHO-K1中已经形成稳定表达。薄层层析（TLC）结果显示,含有不同HCV基因的稳定传代细胞系中,UDP-葡萄糖神经酰胺葡萄糖基转移酶（UGCG）活性均发生了不同程度的变化,其中E2和p7的表达使胞内UGCG的活性提高了约1倍,NS2和NS5A则使UGCG的酶活提高了约0.6倍。该稳定细胞系的建立为研究病毒与宿主因子的相互作用及药物筛选奠定了基础。     

丙型肝炎病毒功能基因在CHO细胞中的表达研究

丙型肝炎病毒(HCV)与宿主细胞因子的相互作用已经成为国内外研究的热点和难点。近期研究已经证实HCV的感染对宿主多种途径中基因的转录均能产生影响。为了进一步研究究竟是HCV中的哪些功能基因在与特定细胞因子的相互作用中起主导作用,构建了分别含有HCV Core、E1、E2、p7、NS2、NS3、NS4A、NS4B、NS5A和NS5B基因的真核表达质粒,分别转入宿主细胞CHO-K1中,在G418的选择压力下筛选获得稳定表达HCV单个蛋白的细胞系(10株)。PCR和RT-PCR可分别从稳定细胞系中检测到相应的HCV基因的DNA和mRNA,冻存和复苏不会造成HCV基因的丢失。Western-blot检测到稳定细胞系中表达E1,E2和NS5B蛋白,说明HCV基因在CHO-K1中已经形成稳定表达。薄层层析(TLC)结果显示,含有不同HCV基因的稳定传代细胞系中,UDP-葡萄糖神经酰胺葡萄糖基转移酶(UGCG)活性均发生了不同程度的变化,其中E2和p7的表达使胞内UGCG的活性提高了约1倍,NS2和NS5A则使UGCG的酶活提高了约0.6倍。该稳定细胞系的建立为研究病毒与宿主因子的相互作用及药物筛选奠定了基础。     

Genetic Immunization of Wild-Type and Hepatitis C Virus Transgenic Mice Reveals a Hierarchy of Cellular Immune Response and Tolerance Induction against Hepatitis C Virus Structural Proteins

To study the effect of genetic immunization on transgenic expression of hepatitis C virus (HCV) proteins, we evaluated the immunological response of HCV transgenic mice to HCV expression plasmids. FVB/n transgenic mice expressing HCV structural proteins (core, E1, and E2) and wild-type (WT) FVB/n mice were immunized intramuscularly with plasmids expressing core (pHCVcore) or core/E1/E2 (pHCVSt). After immunization, HCV-specific humoral and cellular immune response was studied. Both WT and transgenic mice immunized with either HCV construct produced antibodies and exhibited T-cell proliferative responses against core or envelope. In WT mice immunized with pHCVSt, cytotoxic T-lymphocyte (CTL) activities were detected against E2 but not against core or E1, whereas strong CTL activities against core could be detected in WT mice immunized with pHCVcore. In pHCVSt-immunized, transgenic mice, CTL activities against the core or envelope were completely absent, but core-specific CTL activities could be detected in pHCVcore-immunized transgenic mice. A similar pattern of immune responses was also observed in other mouse strains, including a transgenic line expressing human HLA-A2.1 molecules (AAD mice). Despite the presence of a peripheral cellular immunity against HCV, no liver pathology or lymphocytic infiltrate was observed in these transgenic mice. Our study suggests a hierarchy of CTL response against the HCV structural proteins (E2 > core > E1) in vivo when the proteins are expressed as a polyprotein. The HCV transgenic mice can be induced by DNA immunization to generate anti-HCV antibodies and anticore CTLs. However, they are tolerant at the CTL level against the E2 protein despite DNA immunization.     

不同HCV基因型C蛋白在HepG2细胞引起基因表达改变的初步研究

目的:探讨丙型肝炎病毒(HCV)不同基因型C蛋白在HepG2细胞中的基因表达。方法:分别构建能在HepG2细胞表达HCV-1b、HCV-2a和HCV-4d等3种基因型C蛋白的重组体,将Affymetrix公司人基因芯片HG-U133A和HG-U133B用于本研究。结果:3种C蛋白均可引起不同基因上调和下调改变。3种C蛋白表达如两两相比,有若干相同基因表达改变;如三者相比,有PPM1A、TNNI2、ZNF236、FSCN1基因表达出现相同改变。结论:HCV不同基因型C蛋白所引起的基因表达谱各有特征,主要涉及分子转运、信号转导、致病或癌基因等,这对从基因表达层面认识HCVC蛋白的功能及HCV致病机制均有重大帮助。     

丙型肝炎病毒核心蛋白基因真核表达质粒的构建和表达

目的:构建丙型肝炎病毒(HCV)核心蛋白真核表达质粒,并在HepG2细胞中稳定表达.方法:采用RT-RCR方法从丙型肝炎患者血清中得到HCVC区的cDNA序列,然后克隆入pcDNA3.0载体,构建真核表达质粒pcDNA-HCV,并进行酶切鉴定和测序鉴定;采用脂质体转染技术将pcDNA-HCV稳定转染于HepG2细胞系,并用G-418进行筛选;采用Western Blotting方法和免疫细胞化学方法检测HepG2细胞中HCV核心蛋白表达情况.结果:从HCV感染者血清中扩增得到的503bpHCV cDNA序列.属于HCV 1型基因C区,并成功构建了真核表达质粒pcDNA-HCV.经Western blotting和免疫细胞化学检测,稳定转染的HepG2细胞中有HCV核心蛋白的表达.结论:成功构建HCV核心蛋白真核表达质粒pcDNA-HCV,并可以在真核细胞HepG2中稳定表达.     

A Novel Diagnostic Target in the Hepatitis C Virus Genome

Background

Detection and quantification of hepatitis C virus (HCV) RNA is integral to diagnostic and therapeutic regimens. All molecular assays target the viral 5′-noncoding region (5′-NCR), and all show genotype-dependent variation of sensitivities and viral load results. Non-western HCV genotypes have been under-represented in evaluation studies. An alternative diagnostic target region within the HCV genome could facilitate a new generation of assays.

Methods and Findings

In this study we determined by de novo sequencing that the 3′-X-tail element, characterized significantly later than the rest of the genome, is highly conserved across genotypes. To prove its clinical utility as a molecular diagnostic target, a prototype qualitative and quantitative test was developed and evaluated multicentrically on a large and complete panel of 725 clinical plasma samples, covering HCV genotypes 1–6, from four continents (Germany, UK, Brazil, South Africa, Singapore). To our knowledge, this is the most diversified and comprehensive panel of clinical and genotype specimens used in HCV nucleic acid testing (NAT) validation to date. The lower limit of detection (LOD) was 18.4 IU/ml (95% confidence interval, 15.3–24.1 IU/ml), suggesting applicability in donor blood screening. The upper LOD exceeded 10⁻⁹ IU/ml, facilitating viral load monitoring within a wide dynamic range. In 598 genotyped samples, quantified by Bayer VERSANT 3.0 branched DNA (bDNA), X-tail-based viral loads were highly concordant with bDNA for all genotypes. Correlation coefficients between bDNA and X-tail NAT, for genotypes 1–6, were: 0.92, 0.85, 0.95, 0.91, 0.95, and 0.96, respectively; X-tail-based viral loads deviated by more than 0.5 log10 from 5′-NCR-based viral loads in only 12% of samples (maximum deviation, 0.85 log10). The successful introduction of X-tail NAT in a Brazilian laboratory confirmed the practical stability and robustness of the X-tail-based protocol. The assay was implemented at low reaction costs (US$8.70 per sample), short turnover times (2.5 h for up to 96 samples), and without technical difficulties.

Conclusion

This study indicates a way to fundamentally improve HCV viral load monitoring and infection screening. Our prototype assay can serve as a template for a new generation of viral load assays. Additionally, to our knowledge this study provides the first open protocol to permit industry-grade HCV detection and quantification in resource-limited settings.     

Next-Generation Sequencing Reveals Frequent Opportunities for Exposure to Hepatitis C Virus in Ghana

Globally, hepatitis C Virus (HCV) infection is responsible for a large proportion of persons with liver disease, including cancer. The infection is highly prevalent in sub-Saharan Africa. West Africa was identified as a geographic origin of two HCV genotypes. However, little is known about the genetic composition of HCV populations in many countries of the region. Using conventional and next-generation sequencing (NGS), we identified and genetically characterized 65 HCV strains circulating among HCV-positive blood donors in Kumasi, Ghana. Phylogenetic analysis using consensus sequences derived from 3 genomic regions of the HCV genome, 5''-untranslated region, hypervariable region 1 (HVR1) and NS5B gene, consistently classified the HCV variants (n = 65) into genotypes 1 (HCV-1, 15%) and genotype 2 (HCV-2, 85%). The Ghanaian and West African HCV-2 NS5B sequences were found completely intermixed in the phylogenetic tree, indicating a substantial genetic heterogeneity of HCV-2 in Ghana. Analysis of HVR1 sequences from intra-host HCV variants obtained by NGS showed that three donors were infected with >1 HCV strain, including infections with 2 genotypes. Two other donors share an HCV strain, indicating HCV transmission between them. The HCV-2 strain sampled from one donor was replaced with another HCV-2 strain after only 2 months of observation, indicating rapid strain switching. Bayesian analysis estimated that the HCV-2 strains in Ghana were expanding since the 16^th century. The blood donors in Kumasi, Ghana, are infected with a very heterogeneous HCV population of HCV-1 and HCV-2, with HCV-2 being prevalent. The detection of three cases of co- or super-infections and transmission linkage between 2 cases suggests frequent opportunities for HCV exposure among the blood donors and is consistent with the reported high HCV prevalence. The conditions for effective HCV-2 transmission existed for ~ 3–4 centuries, indicating a long epidemic history of HCV-2 in Ghana.     

Characterization of Hepatitis C Virus Intra- and Intergenotypic Chimeras Reveals a Role of the Glycoproteins in Virus Envelopment

Hepatitis C virus (HCV) is highly variable and associated with chronic liver disease. Viral isolates are grouped into seven genotypes (GTs). Accumulating evidence indicates that viral determinants in the core to NS2 proteins modulate the efficiency of virus production. However, the role of the glycoproteins E1 and E2 in this process is currently poorly defined. Therefore, we constructed chimeric viral genomes to explore the role of E1 and E2 in HCV assembly. Comparison of the kinetics and efficiency of particle production by intragenotypic chimeras highlighted core and p7 as crucial determinants for efficient virion release. Glycoprotein sequences, however, had only a minimal impact on this process. In contrast, in the context of intergenotypic HCV chimeras, HCV assembly was profoundly influenced by glycoprotein genes. On the one hand, insertion of GT1a-derived (H77) E1-E2 sequences into a chimeric GT2a virus (Jc1) strongly suppressed virus production. On the other hand, replacement of H77 glycoproteins within the GT1a-GT2a chimeric genome H77/C3 by GT2a-derived (Jc1) E1-E2 increased infectious particle production. Thus, within intergenotypic chimeras, glycoprotein features strongly modulate virus production. Replacement of Jc1 glycoprotein genes by H77-derived E1-E2 did not grossly affect subcellular localization of core, E2, and NS2. However, it caused an accumulation of nonenveloped core protein and increased abundance of nonenveloped core protein structures with slow sedimentation. These findings reveal an important role for the HCV glycoproteins E1 and E2 in membrane envelopment, which likely depends on a genotype-specific interplay with additional viral factors.     

丙型肝炎病毒核心蛋白基因表达致人肝细胞恶性转化的研究

丙型肝炎病毒核心（HCV-C）蛋白是维持丙型肝炎病毒结构的重要蛋白质,由于参与调节细胞的生长与凋亡,被认为与HCV感染所致的肝硬化及肝细胞癌的发生有关.为了进一步探索HCV-C蛋白与肝细胞癌发生的关系,首先构建了表达HCV-C蛋白的真核表达载体,脂质体介导转染Chang-liver人肝细胞株,建立表达HCV-C蛋白的人肝细胞模型,RT-PCR方法检测HCV-C基因在人肝细胞内的表达,蛋白质印迹和免疫细胞化学方法鉴定Chang-liver肝细胞内HCV-C蛋白及其在细胞内的分布情况.表达HCV-C蛋白的Chang-liver人肝细胞培养20代以后,与对照组细胞相比,细胞的形态出现长梭形样改变,生长速度显著加快,细胞内DNA含量的均一性变差.接种表达HCV-C蛋白的Chang-liver人肝细胞的6只裸鼠在第20天时全部有肿瘤长出,且肿瘤组织结构符合肝细胞癌病理形态特点,对照组裸鼠未见肿瘤生长.上述结果表明HCV-C基因表达可导致Chang-liver人肝细胞发生恶性转化,提示HCV-C蛋白与HCV感染所致肝细胞癌的发生有直接关系.     

丙型肝炎病毒RNA多聚酶在昆虫细胞中的表达

ＨＣＶ ＮＳ５Ｂ基因片段克隆入ＢＡＣ－ＴＯ－ＢＡＣ＾ＴＭ重组杆状病毒表达系统的ｐＦＡＳＴＨＴｃ载体质粒,转化ＤＨ１０ＢＡＣ＾ＴＭ感受态细菌获得重组的Ｂａｃｍｉｄ质粒,将重组Ｂａｃｍｉｄ质粒转染Ｓｆ细胞,获得的重组杆状病毒可表达目的蛋白。免疫印迹和体外活性检测表明,所表达蛋白为ＨＣＶ ＮＳ５Ｂ蛋白,具有多聚酶活性。     

丙型肝炎病毒RNA多聚酶在昆虫细胞中的表达

HCV NS5B基因片段克隆入BAC-TO-BACTM重组杆状病毒表达系统的pFASTHTc载体质粒,转化DH10BACTM感受态细菌获得重组的Bacmid质粒,将重组Bacmid质粒转染Sf9细胞,获得的重组杆状病毒可表达目的蛋白.免疫印迹和体外活性检测表明,所表达蛋白为HCV NS5B蛋白,具有多聚酶活性.     

鸭乙型肝炎病毒基因组结构及其编码蛋白的研究进展

鸭乙型肝炎病毒(DHBV)属于禽嗜肝病毒属、嗜肝DNA病毒科病毒,与人乙型肝炎病毒(HBV)具有相同的复制方式及相似的基因结构、抗原结构。DHBV基因组是闭合环状不完全双链DNA,3个主要ORF-PreS/S、ORF-PreC/C、ORF-P位于负链,编码L蛋白、S蛋白、C蛋白和P蛋白。本文通过对DHBV基因组结构特征及其编码病毒重要蛋白的基本特性、结构特点与功能进行全面阐释,旨在对DHBV的深入研究提供重要参考和以DHBV人工感染建立的动物模型研究HBV提供重要理论依据。     

HCV/HBV复合抗原基因的克隆、表达及免疫应答研究

为探讨ＨＣＶ／ＨＢＶ 复合疫苗的可行性,将合成的丙型肝炎病毒（ＨＣＶ）复合多表位抗原基因ＰＣＸ与ＨＢｓＡｇ 基因连接成ＰＣＸＳ基因,与β－半乳糖苷酶（ＧＺ）基因融合后在大肠杆菌及减毒鼠伤寒沙门氏菌中获得表达．目的蛋白ＧＺ－ＰＣＸＳ可被抗－ＨＢｓ 及抗－ＨＣＶ 抗体所特异识别．ＧＺ－ＰＣＸＳ抗原皮下注射免疫ＩＣＲ小鼠后,诱发了较高水平的抗－ＧＺ－ＰＣＸＳＩｇＧ反应．构建的重组减毒鼠伤寒沙门氏菌ＳＬ３２６１（ｐＷＲ／ＰＣＸＳ）口服免疫小鼠后,诱发了高水平的ＣＤ８＋ Ｔ细胞增殖反应及抗ＧＺ－ＰＣＸＳＩｇＧ反应．所有免疫小鼠均未见明显的毒副作用．该研究揭示,ＨＣＶ／ＨＢＶ 复合抗原可诱发特异性体液免疫及细胞免疫应答,而活菌苗口服可能是理想的免疫途径,为ＨＣＶ／ＨＢＶ 双价疫苗研究提供了一定的理论及实验依据．     

Liver Fibrosis,Host Genetic and Hepatitis C Virus Related Parameters as Predictive Factors of Response to Therapy against Hepatitis C Virus in HIV/HCV Coinfected Patients

Objective

To establish the role of liver fibrosis as a predictive tool of response to pegylated interferon alpha (Peg-IFN) and ribavirin (RBV) treatment in human immunodeficiency (HIV)/hepatitis C virus (HCV) coinfected patients, in addition to recognized predictive factors (HCV load, HCV genotype, IL-28B polymorphism).

Patients and Methods

A sample of 267 HIV/HCV coinfected patients was treated with Peg-IFN and RBV. Predictive factors of rapid (RVR) and sustained (SVR) virological response were analyzed. Independent variables were age, sex, IL28B, −238 TNF-α and −592 IL-10 polymorphisms, HCV genotype, HCV-RNA levels, significant fibrosis or cirrhosis and CD4+ T cell count.

Results

Patients infected by HCV genotype 1 (n = 187) showed RVR and SVR in 12% and 39% of cases, respectively. The parameters associated with RVR were IL28B genotype CC and plasma HCV-RNA levels <600000 IU/ml. Advanced liver fibrosis was negatively associated with SVR in patients without RVR. A SVR was obtained in 42% of subjects with HCV genotype 4, and the independent factors associated with SVR were IL28B genotype CC and an HCV-RNA <600000 IU/ml. A SVR was obtained in 66% of patients with HCV genotypes 2/3; in this case, the independent parameter associated with SVR was the absence of significant liver fibrosis. TNF-α and IL-10 polymorphisms were not associated with SVR, although a significantly higher percentage of −238 TNF-α genotype GG was detected in patients with significant liver fibrosis.

Conclusions

In HIV/HCV coinfected patients with HCV genotypes 1 or 4, RVR, mainly influenced by genotype IL28B and HCV-RNA levels, reliably predicted SVR after 4 weeks of therapy with Peg-IFN plus RBV. In patients infected by HCV genotype 3, an elevated relapse rate compromised the influence of RVR on SVR. Relapses were related to the presence of advanced liver fibrosis. Liver cirrhosis was associated with a −238 TNF-α polymorphism in these patients.     

乙肝病毒表面抗原preS1与人肿瘤坏死因子α融合基因的表达

用ＰＣＲ法获得了ＨＢｓＡｇｐｒｅＳ１（１－６５）肽段基因,将该基因融合在肿瘤坏死因子（ｈＴＮＦα）之后,插入表达载体ＰＳＢ－９２中,使融合基因的５′端直接置于大肠肝菌ＰＬ启动子下游,采用３０℃培养,４２℃诱导,获得了ＴＮＦ与ｐｒｅＳ１（１－６５）融合蛋白的表达产物。ＳＤＳ－ＰＡＧＥ电泳显示表达产物为２５ｋＤ,约占细菌总蛋白的３５％。表达产物经Ｗｅｓｔｅｒｎｂｌｏｔ验证,能分别特异地与ｈＴＮＦα抗体与ｐｒｅＳ１抗体结合,稀释复性后,该融合蛋白还具有ＴＮＦ的生理功能（对Ｌ９２９细胞的细胞毒活性）。经ＤＮＡ序列测定,ｐｒｅＳ１（１－６５）肽基因正确地融合在ｈＴＮＦα基因之后。该结果提供了一种制备ｐｒｅＳ１的新方法,为进一步开展治疗肝癌和乙肝的导向药物打下基础。     

Identification of RNA Binding Proteins Associated with Dengue Virus RNA in Infected Cells Reveals Temporally Distinct Host Factor Requirements

BackgroundThere are currently no vaccines or antivirals available for dengue virus infection, which can cause dengue hemorrhagic fever and death. A better understanding of the host pathogen interaction is required to develop effective therapies to treat DENV. In particular, very little is known about how cellular RNA binding proteins interact with viral RNAs. RNAs within cells are not naked; rather they are coated with proteins that affect localization, stability, translation and (for viruses) replication.Conclusions/SignificanceThe method for identification of host factors described here is robust and broadly applicable to all RNA viruses, providing an avenue to determine the conserved or distinct mechanisms through which diverse viruses manage the viral RNA within cells. This study significantly increases the number of cellular factors known to interact with DENV and reveals how DENV modulates and usurps cellular proteins for efficient amplification.