丙型肝炎病毒不同基因型NS3蛋白的抗原异质性分析

目的探讨不同基因型丙型肝炎病毒(HCV)NS3蛋白的抗原特性及其用于抗-HCV检测的意义。方法分别构建和表达含有HCV1型和6型NS3基因片段的重组质粒和重组蛋白，以EIJSA法和Western blot分析不同基因型重组蛋白与已知抗-HCV阳性血清的抗原反应性。结果HCV1型和6型NS3重组蛋白氨基酸序列的同源性为83．2％；85份抗-HCV阳性血清以此不同基因型HCV NS3单片段抗原检测，阳性检出率分别为61．2％(1型)和58．8％(6型)，其中有7份标本以NS3-1型抗原检测阴性，但可被NS3-6型抗原检出，反之，有9份血清以NS3-6型重组蛋白检测为阴性，而NS3．1型检测阳性；54份大学生体检血清和39份阴性质控血清以此两种抗原检测均为阴性。结论HCV1型和6型NS3重组蛋白存在抗原异质性，在发展HCV抗体检测试剂时需考虑加入不同基因型的NS3抗原。     

丙型肝炎病毒NS5a高免疫原区人工合成多肽抗原性的研究

目的 检测不同基因型丙型肝炎病毒(HCV)非结构5a区(NS5a)高免疫原区短链多肽的抗原性，确定该区的主要线性抗原表位并探讨基因异质性与免疫原性间的关系。方法 设计并合成特异性多肽；DNA Star软件对多肽氨基酸同源性进行比较；间接ELISA法检测多肽的抗原性。结果多肽氨基酸的同源性在不同区及不同基因型间变化较大。氨基酸残基2212～2241、2272-2301和2302-2331的多肽抗原性最强。18个来自保守区的多肽可以与不同基因型抗-HCV阳性血清起反应(阳性率高达96％)；而来自不同基因型间氨基酸序列保守性较低区的多肽抗原性与同基因血清的反应性较高。结论 HCV NS5a高免疫原区主要的线性抗原位于氨基酸残基2212-2241、2272-2301和2302-2331的位置；人工合成的多肽可以有效地用于检测抗-HCV抗体；一些多肽的抗原性具有基因型特异性。     

Antigenic structure of the hepatitis C virus envelope 2 protein.

The antigenic structure of the envelope 2 (e2) protein of the hepatitis C virus (HCV) was characterized by the use of 70 synthetic peptides and 131 human sera from persons with antibodies to HCV. Among 34 overlapping peptides spanning the e2 protein of HCV, two major antigenic regions were located to residues 484-499 and residues 554-569. The sequence of the two major antigenic regions of the e2 protein are partly well conserved within the described types of HCV. Both regions contain two Cys residues in close proximity, and the region at residues 554-569 contains a putative N-glycosylation site, which are factors that previously have been suggested to affect the immune recognition of the e2 protein. Using substitution peptide analogues where each position within residues 484-499 and 554-569 were sequentially substituted by Ala or Gly, the most essential residues for antibody binding were found to be the Pro-498, Ala-499, Ala-566, Pro-567, and Pro-568. All of these, except for the Pro-498 and Ala-566, are conserved among different HCV strains. Also, according to previous studies, position 496 often shows variations, which could be explained by position 496 being contained within the antigenic region at residues 484-499. Interestingly, none of the Cys residues at positions 486, 494, 564 and 569 were found to be essential for antibody binding, indicating that these are not essential in maintaining the e2 antigenicity of the peptides. In a material of 114 confirmed anti-HCV positive sera, derived from patients during the acute or the chronic phase of HCV infection, the prevalence of antibodies to the two major linear antigenic regions of the e2 protein was found to be 55% among HCV RNA-positive sera, and 53% among HCV RNA-negative sera. In conclusion, we have identified and characterized two major linear antigenic regions outside the two hypervariable regions of the e2 protein. Since these regions are accessible to the B cells of the infected host, these two regions are likely to be surface exposed either on the precursor polyprotein or the native e2 protein. Also, we could confirm that antibodies to the e2 protein co-exist with HCV viraemia.     

用合成寡肽的酶免疫法检测丙型肝炎病毒血清型

目的探讨适合我国临床广泛推广应用的丙型肝炎分型方法。方法采用丙型肝炎病毒核心区及ＮＳ４区合成寡肽建立酶联免疫检测方法，对血清中丙型肝炎病毒抗体进行分型。用此方法对某地个体供血者及慢性肝病患者的血清标本进行测定。首先使用美国ＡｂｂｏｔＩＭｘ试剂测定抗－ＨＣＶ，阳性者再测定其血清型。为验证血清分型与基因型的关系，对ＨＣＶＲＮＡ阳性的标本用型特异性引物的分型ＰＣＲ方法测定其基因型。结果７０例已往单采血浆供血者中ＨＣＶＲＮＡ阳性为３０％，抗－ＨＣＶ阳性率６８．６％，其中血清分型阳性率为７２．９％。血清１型与基因Ⅱ（１ｂ）型相对应；血清２型与基因Ⅲ（２ａ）型相对应。结论本方法操作简便、快速，具有较强特异性，基本能满足国内临床需要，对丙型肝炎发病及流行病学研究具有重要意义。     

Detection of type specific antibodies to hepatitis C virus NS4 protein in hepatitis C patients by enzyme immunoassay

A multi-enzyme immmune-assay test system was designed for serotyping of genotypes hepatitis C virus (HCV) and a method of such typing of the serum of patients with hepatitis C was worked out. The above test-system was worked out on the basis of a study of 10 type-specific peptides modeling different fragments from NS4-protein variable region of HCV. The designed test system was evaluated by using a set of 42 serum samples obtained at random from patients with chronic hepatitis C, which had been preliminarily genotyped by polymerase chain reaction. The serotyping makes it possible to identify the type-specific antibodies in the blood sera of patients, including those cases when viremia was absent. Differences in the circulation of HCV in Moscow (Russia) and Vitebsk (Byelorussia) were established by using the designed test-system.     

Characterization of monoclonal antibodies and epitope mapping of the NS4 protein of hepatitis C virus

Recombinant DNA containing sequences of HCV NS4 protein was expressed in Escherichia coli cells. Six hybridoma clones producing monoclonal antibodies (MAB) to recombinant NS4 protein (rNS4), aa 1677-1756, were developed. Mapping with a panel of 33 peptides and reciprocal competitive EIA have shown that MAB obtained revealed five antigen determinants, not described earlier: MAB 3F11 and 3F12-one genotype-independent epitope of NS4A (aa 1700-1707) common for genotypes 1, 2 and 3; MAB 1D11-genotype-independent epitope (aa 1713-1728) and MAB 1D3-genotype (subtype 1b)-specific epitope of NS4B (aa 1711-1731); MAB 6B11 and C1-two conformation-dependent determinants in 5-1-1 region. These data indicate that the 5-1-1 region of NS4 protein has a complex antigenic structure and contains at least eight epitopes, including five, revealed in the present work. MAB obtained recognized native viral protein in the cytoplasm of liver cells of patients with chronic hepatitis C. The positive rates of the immunostaining for NS4 antigen using MAB 6B11, 1D11 and 3F12 were 64, 59 and 50%, respectively. It was found that 6B11 MAB to a conformation-dependent epitope much more actively interacts with native NS4 than with the recombinant protein to which MAB was developed. The epitope recognized by 6B11 MAB is highly immunogenic since it induces the B-cell response in all patients investigated with identified anti-NS4 antibodies in blood serum. The MAB panel obtained in this study may become a useful tool for the diagnostic purposes, for the investigation of NS4B function and for the host-viral interactions at the cell level.     

Artificial NS4 mosaic antigen of hepatitis C virus.

An artificial antigen composed of 17 small antigenic regions derived from the NS4-protein of hepatitis C virus (HCV) genotypes 1 through 5 was designed and constructed. Eleven antigenic regions were derived from the 5-1-1 region, and 6 others were derived from the C-terminus of the NS4-protein of different genotypes. The gene encoding for this artificial antigen was assembled from synthetic oligonucleotides by a new approach designated as restriction enzyme-assisted ligation (REAL). The full-length synthetic gene was expressed in Escherichia coli as a fusion protein with glutathione S-transferase. By the use of site-specific antibodies raised against synthetic peptides, it was shown that all regions for which sequence-specific antibodies were obtained were accessible to antibody binding. The diagnostic relevance of the NS4 artificial antigen was demonstrated by testing this antigen with 4 HCV seroconversion panels and a panel of previously tested and stored serum specimens. The artificial antigen was found to specifically detect anti-NS4 antibodies in a number of specimens that were previously found to be anti-NS4 negative. Furthermore, this antigen detected anti-NS4 activity earlier in 2 of 4 seroconversion panels than did the antigen used in a commercially available supplemental assay. Equally important is the observation that the artificial NS4 antigen demonstrated equivalent anti-NS4 immunoreactivity with serum specimens obtained from patients infected with different HCV genotypes, whereas the NS4 recombinant protein derived from genotype 1, used in the commercial supplemental test, was less immunoreactive with serum specimens containing HCV genotypes 2, 3, and 4. Collectively, these data support the significant diagnostic potential of the NS4 mosaic antigen. The strategy employed in this study may be applied to the design and construction of other artificial antigens with improved diagnostically pertinent properties. J. Med. Virol. 59:437-450 1999.     

Antigenic variation of core, NS3, and NS5 proteins among genotypes of hepatitis C virus.

Assays that detect antibody to hepatitis C virus (HCV) are used to screen blood donors and patients with hepatitis. Current enzyme-linked immunosorbent assay (ELISA)-based methods are invariably based upon antigens from expressed recombinant proteins or oligopeptides from HCV type 1. Some HCV antigens used in screening assays are coded by regions of the HCV genome that show extensive variability; therefore, HCV type 1-based assays may be less effective for the detection of antibody elicited by infection with other genotypes. In this study, we have measured antibody reactivity of sera from 110 hepatitis C patients infected with type 1b, 3a, or 4a to genotype-specific and cross-reactive epitopes present in recombinant proteins from HCV genotypes 1b (core, NS3, and NS5), 3a (NS3, NS5), and 4a (core, NS3), corresponding to those used in current third-generation screening ELISAs. By comparing the serological reactivities of sera to type-homologous and type-heterologous antigens, we detected a significant type-specific component to the reactivity to NS3 (61 to 77% of the total reactivity) and NS5 (60% of the total reactivity). Furthermore, despite the similarities in the amino acid sequences of the core antigens of type 1b and type 4a, we also found significantly greater reactivity to type-homologous antigens, with approximately 25% of reactivity being type specific. These findings are consistent with previous findings of fivefold weaker reactivity of sera from HCV type 2- and HCV type 3-infected blood donors in the currently used third-generation ELISAs and suggest that these assays are suboptimal for screening populations in which the predominant genotype is not type 1.     

丙型肝炎病毒非结构蛋白NS3对端粒酶活性的影响

目的研究丙型肝炎病毒非结构区3(HCV NS3)蛋白对端粒酶活性的影响,以探讨HCV NS3蛋白在HCV致癌中的作用,并观察端粒酶活性原位检测法的应用价值.方法利用HCV NS3真核细胞表达质粒pRcHCNS3-5′(表达HCV NS3 N端多肽),pRcHCNS3-3′(表达HCV NS3C端多肽)和空白质粒pRcCMV转染NIH3T3细胞,分别获得11、11和8个阳性克隆;采用链霉素抗生物素-过氧化物酶法(SP)免疫组织化学方法检测转染的NIH3T3细胞中HCV NS3蛋白表达,并通过端粒酶活性原位检测法和端粒酶聚合酶链反应(PCR)酶联免疫吸附反应(ELISA)技术分别检测转染前后NIH3T3细胞端粒酶活性的定位和定量变化.结果 HCV NS3表达质粒pRcHCNS3-5′或pRcHCNS3-3′转染的NIH3T3细胞均表达HCV NS3蛋白,HCV NS3蛋白阳性信号均位于细胞质中,并以前者表达的阳性信号为强(χ2=6.667,P<0.05),各组细胞端粒酶活性存在显著差异(F=143.083,P<0.01),其中质粒pRcHCNS3-5′转染的NIH3T3细胞端粒酶活性最强,11个克隆均呈阳性,质粒pRcHCNS3-3′转染的细胞次之(P<0.05),空白质粒pRcCMV转染细胞和未转染NIH3T3细胞最弱;HCV NS3蛋白的表达水平和端粒酶活性强度之间具有显著相关性(rs=0.808 4,P<0.01);采用端粒酶活性原位检测方法和端粒酶PCR ELISA技术检测结果具有较好的一致性(rs=0.501 96,P<0.01).结论 (1) HCV NS3蛋白可能是通过内源性机制激活细胞端粒酶导致宿主细胞恶性转化;(2) HCV NS3蛋白 N端多肽对宿主细胞端粒酶的激活作用强于C端多肽;(3) 进一步证实端粒酶活性原位检测法是一种适合于病理形态与功能研究的技术.     

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.     

Restricted isotypic antibody reactivity to hepatitis C virus synthetic peptides in immunocompromised patients

An enzyme immunoassay based on three synthetic peptides from the core, NS4, and NS5 regions of hepatitis C virus allowed the detection of antibodies in 100% of immunocompetent infected patients and in 91% of immunocompromised patients (hemodialysis and hemophiliac patients). Immune impairment seemed to restrict the spectrum of antibody isotypes reacting to the core peptide.     

Antigenic variation in the influenza A virus nonstructural protein, NS1

The antigenic structure of the nonstructural (NS1) protein encoded by influenza type A virus was examined using monoclonal antibodies prepared against purified NS1 inclusions isolated from the cytoplasm of infected cells. Topographical analysis by competitive radioimmunoassay indicated that three different overlapping antigenic regions were present on the NS1 of A/WSN/33 (H1N1). Immunoprecipitation studies using infected cell lysates showed that antigenic determinants on A/WSN/33 NS1 are common to NS1 proteins encoded by a wide range of viruses of human, swine, equine, and avian origin. Several avian strains, however, were found to encode antigenically variant NS1 proteins which had either extensive changes in one or more antigenic regions or small changes in epitopes within a region suggestive of antigenic drift. There was no correlation between surface antigen subtype and the antigenic profile of the NS1 protein. The antigenic relationships of NS1 proteins shown in this study are in agreement with the available sequence data.     

Internal cleavage of hepatitis C virus NS3 protein is dependent on the activity of NS34A protease

The nonstructural protein NS3 of the hepatitis C virus (HCV) is indispensable for virus replication and a multifunctional enzyme that contains three catalytic activities such as serine protease, helicase, and NTPase. Here, we demonstrated that the internal cleavage of the HCV NS3 protein occurs in various mammalian cells such as HepG2, COS-7, and NIH3T3. As is observed for the internal cleavage mechanism of the NS3 protein of dengue virus 2, the internal processing of HCV NS3 protein was catalyzed by the active NS3 serine protease and NS4A, but not NS3 alone. From the data acquired from extensive site-directed mutagenesis, we observed that the NS3 protein was internally cleaved at two different sites, FCH(1395) ||S(1396)KK and IPT(1428) ||S(1429)GD, within RNA helicase domain. The internal cleavage of NS3 protein by NS34A protease was also confirmed in a different isolate of HCV-1b strain. In addition, in vitro transforming assays demonstrated that the internal cleavage product of NS3, NS3a-1, appeared to have higher oncogenic potential than does intact NS3. Taken together, our results suggest that the internal cleavage of NS3 may be associated with the replication and oncogenesis of HCV.     

Genetic heterogeneity of the NS3 protease gene in hepatitis C virus genotype 1 from untreated infected patients

NS3 protease is essential for hepatitis C Virus (HCV) replication, and is one of the most promising targets for specific anti-HCV therapy. Its natural polymorphism has not been studied at the quasispecies level. In the present work, the genetic heterogeneity of the NS3 protease gene was analyzed in 17 HCV genotype 1 (5 subtypes 1a and 12 subtypes 1b) samples collected from infected patients before anti-viral therapy. A total of 294 clones were sequenced. Although the protease NS3 is considered to be one of the less variable genes in the HCV genome, variability of both nucleotide and amino acid sequences was found. In variants belonging to 1a and 1b subtypes, 224 and 267 of 543 positions showed one or more nucleotide substitutions, respectively. Forty and 74 of the 181 NS3 amino acid positions showed at least one mutation in HCV-1a and HCV-1b isolates, respectively. Most substitutions were conservative. This substantial polymorphism of the NS3 protease produced by HCV-1a and HCV-1b suggests that, despite the numerous functional and structural constraints, the enzyme is sufficiently flexible to tolerate substitutions.     

Formation and function of hepatitis C virus replication complexes require residues in the carboxy-terminal domain of NS4B protein

During replication, hepatitis C virus (HCV) NS4B protein rearranges intracellular membranes to form foci, or the web, the putative site for HCV replication. To understand the role of the C-terminal domain (CTD) in NS4B function, mutations were introduced into NS4B alone or in the context of HCV polyprotein. First, we show that the CTD is required for NS4B-induced web structure, but it is not sufficient to form the web nor is it required for NS4B membrane association. Interestingly, all the mutations introduced into the CTD impeded HCV genome replication, but only two resulted in a disruption of NS4B foci. Further, we found that NS4B interacts with NS3 and NS5A, and that mutations causing NS4B mislocalization have a similar effect on these proteins. Finally, we show that the redistribution of Rab5 to NS4B foci requires an intact CTD, suggesting that Rab5 facilitates NS4B foci formation through interaction with the CTD.     

Complex formation between hepatitis C virus NS2 and NS3 proteins

Hepatitis C virus (HCV) NS2 and NS3 proteins as well as the NS3 protease cofactor NS4A are essential for the replication of the virus. The presence of in vivo heterodimeric complex between HCV NS2 and NS3 has been suggested by biochemical studies. Detailed characterization of the interactions between these viral proteins is of great importance for better understanding their role in viral replication cycle and represents attractive target for antiviral agents. In this study, we demonstrated in vivo interactions between HCV NS2 and NS3 proteins using an epitope tagging technique. For this purpose NS2, NS3 and NS4A were expressed in fusion with two different tags in Cos7 cells. Immunofluorescence analysis and co-immunoprecipitation with tag-specific antibodies revealed the existence of biologically important NS3/NS4A and NS3/NS2 complexes. Similar complexes were detected also in Huh7 cells infected with Semliki Forest virus vectors expressing NS2 and NS3 or NS23 precursor polyprotein. The formation of complex between NS2 and NS3 was found not to depend on whether the proteins were expressed individually or in form of common precursor. This observation suggests the existence of direct interaction between these two proteins that may have importance for the formation of the whole HCV replication complex.     

DNA vaccination protects mice against challenge with Listeria monocytogenes expressing the hepatitis C virus NS3 protein

The goal of this study was to develop a new surrogate challenge model for use in evaluating protective cell-mediated immune responses against hepatitis C virus (HCV) antigens. The use of recombinant Listeria monocytogenes organisms which express HCV antigens provides novel tools with which to assay such in vivo protection, as expression of immunity against this hepatotropic bacterial pathogen is dependent on antigen-specific CD8(+) T lymphocytes. A plasmid DNA vaccine encoding a ubiquitin-NS3 fusion protein was generated, and its efficacy was confirmed by in vivo induction of NS3-specific, gamma interferon-secreting T cells following vaccination of BALB/c mice. These immunized mice also exhibited specific in vivo protection against subsequent challenge with a recombinant L. monocytogenes strain (TC-LNS3) expressing the NS3 protein. Notably, sublethal infection of naive mice with strain TC-LNS3 induced similar NS3-specific T-cell responses. These findings suggest that recombinant strains of L. monocytogenes expressing HCV antigens should prove useful for evaluating, or even inducing, protective immune responses against HCV antigens.     

Antigenic analysis of bean pod mottle virus using linear and cyclized synthetic peptides

Summary The antigenic structure of the comovirus bean pod mottle virus (BPMV) was studied using synthetic peptides selected on the basis of the exposed location of certain regions of the viral protein. Three regions of domain A, four regions of domain B and two regions of domain C of BPMV coat protein were studied. Each of four regions were synthesized in the form of linear and cyclized peptides while the others were synthesized as linear peptides only. The peptides were tested for their ability to be recognized by antibodies directed against BPMV. The peptides were also used for producing rabbit antisera, which were tested for their ability to react with various BPMV antigens as well as with the linear and cyclized peptides. All the peptides were found to correspond to epitopes of BPMV coat protein. Several of the antigenic sites of BPMV located on exposed loops of the coat protein occupy positions which correspond to known epitopes in the structurally related picornaviruses. Only in some cases did cyclization sufficiently improve the level of conformational mimicry between peptides and the viral protein to allow cross-reactions between them to be observed.     

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。