用噬菌体展示随机12肽库筛选HCV B细胞抗原表位

目的用患者血清中抗丙型肝炎病毒(HCV)抗体从噬菌体展示随机12肽库筛选HCV抗原表位。方法用硫酸铵粗提HCV患者血清中Ig后用DEAE—Sephadex A50层析纯化并作为筛选的配基;对噬菌体展示随机12肽库采用配基亲和富集、阴性血清吸收的方法进行生物淘洗;ELISA鉴定筛选克隆的结合特性;测定阳性克隆所携带DNA序列并进行计算机辅助分析。结果三轮生物淘洗后特异性克隆得到了富集。用第3轮淘洗出的26个克隆进行结合实验,其中有3个克隆与HCV患者血清结合力较高而与正常人血清结合能力较弱;序列分析发现2个序列与HCV蛋白第1082～1093和1954～1965位(1082YHGAGTRTIASP 1093和1954TQLLRRLHQWIS 1965)氨基酸有较高的同源性;计算机辅助分析提示这两个位点具有形成表位的性质。结论获得了两个HCV抗原表位,在HCV感染的检测中具有潜在的应用价值。     

Hepatitis C epitopes from phage-displayed cDNA libraries and improved diagnosis with a chimeric antigen

A novel method for cloning DNase I fragments into bacteriophage display vector fUSE2 was used to create libraries expressing hepatitis C virus (HCV) protein fragments on the phage surface. Selection by panning with a mixture of sera from five HCV-seropositive individuals enabled identification of antigenic determinants in NS3 (amino acids 1,383-1,415), NS4 (amino acids 1, 930-1,938), and NS5 (amino acids 2,088-2,104). The NS3 result is the most accurate location to date of a major conformational determinant that cannot be mimicked by short peptides. Any expressed sequence from the phage library can be excised with Bgl II and cloned directly into the Bgl II site of an appropriate plasmid for bacterial expression. This enables production of chimeric proteins containing multiple antigenic determinants, illustrated by co-expression of the NS4P (amino acids 1,930-1,938) epitope with an NS4N fragment (amino acids 1,644-1,812) containing at least three linear HCV epitopes. When used to screen 35 individual HCV-positive sera by enzyme-linked immunosorbent assay (ELISA), the chimeric antigen detected eight more positives than NS4N alone and gave increased immunoreactivity with others. This approach of identifying antigenic regions by phage display and then co-expressing them as chimeric proteins may be generally applicable to the production of improved diagnostic antigens and recombinant vaccines.     

The hepatitis C virus NS5B RNA-dependent RNA polymerase activity and susceptibility to inhibitors is modulated by metal cations

OBJECTIVES: The aim of this study was to understand the effect of metal cations on the hepatitis C virus (HCV) NS5B in vitro RNA-dependent RNA polymerase (RdRp) activity and its susceptibility to various inhibitors. METHODS: A recombinant full-length HCV NS5B protein was expressed in insect cells and purified to homogeneity. RdRp activity was assessed using standard filtration or polyacrylamide gel-based assays. RESULTS: Efficient inhibition of the HCV NS5B RdRp activity by gliotoxin, as well as by various substrate analogs, occurs in the presence of Mn2+, but not of Mg2+. Assays performed in the presence of both cofactors suggest that, in vitro, the enzyme's affinity for Mn2+ is higher than that for Mg2+. In addition, the RdRp activity, displayed in the presence of heteropolymeric templates, is significantly increased when the metal cofactor consists of Mn2+. Finally, steady state kinetics showed that the velocity of the reaction, as well as the affinity of the enzyme for its substrate, could both be affected by the nature of the divalent metal cation used.     

Characterization of mimotopes mimicking an immunodominant conformational epitope on the hepatitis C virus NS3 helicase

The hepatitis C virus (HCV) nonstructural 3 (NS3) protein is composed of an amino terminal protease and a carboxyl terminal RNA helicase. NS3 contains major antigenic epitopes. The antibody response to NS3 appears early in the course of infection and is focused on the helicase region. However, this response cannot be defined by short synthetic peptides indicating the recognition of conformation-dependent epitopes. In this study, we have screened a dodecapeptide library displayed on phage with anti-NS3 mouse monoclonal antibodies (mAbs) that compete with each other and human anti-HCV NS3 positive sera. Two peptides (mimotopes) were selected that appeared to mimic an immunodominant epitope since they were recognized specifically by the different anti-NS3 mAbs of the study and by human sera from HCV infected patients. Homology search between the two mimotopes and the NS3 sequence showed that one of the two peptides shared amino acid similarities with NS3 at residues 1396-1398 on a very accessible loop as visualized on the three-dimensional structure of the helicase domain whereas the other one had two amino acids similar to nearby residues 1376 and 1378. Reproduced as synthetic dodecapeptides, the two mimotopes were recognized specifically by 19 and 22, respectively, out of 49 sera from HCV infected patients. These mimotopes allowed also the detection of anti-NS3 antibodies in sera of HCV patients at the seroconversion stage. These results suggest that the two NS3 mimotopes are potential tools for the diagnosis of HCV infection.     

Characterization of monoclonal antibodies that specifically recognize the palm subdomain of hepatitis C virus nonstructural protein 5B polymerase

The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp) which plays an essential role in viral RNA replication. Antibodies that specifically recognize NS5B will have utilities in monitoring NS5B production and subcellular localization, as well as in structure-function studies. In this report, three mouse monoclonal antibodies (mAbs), 16A9C9, 16D9A4 and 20A12C7, against a recombinant NS5B protein (genotype 1a, H-77 strain) were produced. These mAbs specifically recognize HCV NS5B, but not RdRps of polivirus (PV), bovine viral diarrhea virus (BVDV) or GB virus B (GBV-B). The mAbs can readily detect NS5B in cellular lysates of human osteosarcoma Saos2 cells constitutively expressing the nonstructural region of HCV (NS3-NS4A-NS4B-NS5A-NS5B). NS5B proteins of different HCV genotypes/subtypes (1a, 1b, 2a, 2c, 5a) showed varied affinity for these mAbs. Interestingly, the epitopes for the mAbs were mapped to the palm subdomain (amino acid 188-370) of the HCV RdRp as determined by immunoblotting analysis of a panel of HCV/GBV-B chimeric NS5B proteins. The binding site was mapped between amino acid 231 and 267 of NS5B for 16A9C9, and between 282 and 372 for 16D9A4 and 20A12C7. Furthermore, these mAbs showed no inhibitory effect on the NS5B polymerase activity in vitro.     

Enzymatic activities of the GB virus-B RNA-dependent RNA polymerase

The GB virus-B (GBV-B) nonstructural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase (RdRp) with greater than 50% sequence similarity to the hepatitis C virus (HCV) NS5B. Recombinant GBV-B NS5B was reported to possess RdRp activity (W. Zhong et al., 2000, J. Viral Hepat. 7, 335-342). In this study, the GBV-B RdRp was examined more thoroughly for different RNA synthesis activities, including primer-extension, de novo initiation, template switch, terminal nucleotide addition, and template specificity. The results can be compared with previous characterizations of the HCV RdRp. The two RdRps share similarities in terms of metal ion and template preference, the abilities to add nontemplated nucleotides, perform both de novo initiation and extension from a primer, and switch templates. However, several differences in RNA synthesis between the GBV-B and HCV RdRps were observed, including (i) optimal temperatures for activity, (ii) ranges of Mn(2+) concentration tolerated for activity, and (iii) cation requirements for de novo RNA synthesis and terminal transferase activity. To assess whether the recombinant GBV-B RdRp may represent a relevant surrogate system for testing HCV antiviral agents, two compounds demonstrated to be active at nanomolar concentrations against HCV NS5B were tested on the GBV RdRp. A chain terminating nucleotide analog could prevent RNA synthesis, while a nonnucleoside HCV inhibitor was unable to affect RNA synthesis by the GBV RdRp.     

Expression and purification of enzymatically active recombinant RNA-dependent RNA polymerase (NS5) of the flavivirus Kunjin

The NS5 protein of the flavivirus Kunjin (KUN) contains conserved sequence motifs characteristic of RNA-dependent RNA polymerase (RdRp) activity. To investigate this activity in vitro, recombinant NS5 proteins with C-terminal (NS5CHis) and N-terminal (NS5NHis) hexahistidine tags were produced in baculovirus-infected insect cells and purified to near homogeneity by nickel affinity chromatography. Purified NS5CHis exhibited RdRp activity with both specific (9 kb KUN replicon) and non-specific (8.3 kb Semliki Forest virus replicon) RNA templates; this activity did not require the presence of additional viral and/or cellular cofactors. RdRp activity of purified NS5NHis protein was reduced in comparison to NS5CHis, while purified NS5NHis incorporating a GDD-->GVD mutation within the polymerase active site (NS5GVD) lacked RdRp activity. RNase A digestion of the RdRp reaction products indicated that they were double-stranded and of a similar size to the KUN replicative form produced in Vero cells, thus demonstrating that the KUN NS5 protein has an intrinsic, albeit low and non-specific RdRp activity in vitro, similar to that reported for recombinant RdRp of other flaviviruses. However, in contrast to RNA polymerases of other Flavivirus species, purified KUN NS5 polymerase produced a single, full-length replicon RNA product, thus demonstrating efficient processivity.     

应用噬菌体随机肽库研究丙型肝炎病毒核心蛋白B细胞抗原表位

目的探索利用特异多克隆抗体结合噬菌体递呈（ｐｈａｇｅｄｉｓｐｌａｙ）随机肽库,进行病原体蛋白质的Ｂ细胞抗原表位研究。方法采取特异抗原→特异多克隆抗体→相应抗原表位的技术路线。所选研究对象为丙型肝炎病毒（ＨＣＶ）核心蛋白。实验分三步：（１）抗ＨＣＶ核心蛋白多克隆抗体的制备。用活化的Ｓｅｐｈａｒｏｓｅ４Ｂ耦联重组的ＨＣＶ核心蛋白,亲和层析纯化丙型肝炎病人血清中特异的抗核心蛋白多克隆抗体。（２）随机肽库的生物淘洗（ｂｉｏｐａｎｎｉｎｇ）。以纯化的多克隆抗体作为筛选分子,生物淘洗噬菌体递呈的随机七肽库。（３）阳性噬菌体克隆的鉴定。将筛选的噬菌体克隆进行ＥＬＩＳＡ检测、ＤＮＡ测序以及竞争抑制试验等,最后分析所获资料。结果七肽氨基酸序列分析表明,ＨＣＶ核心蛋白存在着至少３个Ｂ细胞抗原位点,其中１９～２５ａａ间（ＰＱＤＶＫＦＰ）的线性表位是该蛋白的优势表位,证实了本研究策略的可行性。结论本技术路线可以有效地进行ＨＣＶ核心蛋白的Ｂ细胞抗原表位研究。由此推论,此方法也可移植于其它病原微生物抗原或自身抗原的表位研究,继而为基于抗原表位水平的特异诊断试剂的研制、疫苗的设计提供依据     

Characterization of RNA-dependent RNA Polymerase Activity of CSFV NS5B Proteins Expressed in Escherichia coli

The full-length NS5B protein, and the truncated NS5B proteins of classical swine fever virus (CSFV) resulted from deletion of 24, 36, 65 or 82, amino acid residues at the C terminal were expressed in Escherichia coli cells and purified with a C-terminal hexahistidine tag. In addition to the full-length NS5B protein, those truncated NS5B proteins with deletion of 24, 36, or 65 amino acid residues were demonstrated to have RNA-dependent RNA polymerase (RdRp) activity, which was not found in the truncated NS5B proteins with deletion of 82 amino acid residues. Analysis of the template specificity of CSFV RdRp was done containing the different NS5B proteins with RdRp activity. It was shown that the template specificity of the enzyme was not strict with NS5B proteins truncated, suggesting that the C terminal of CSFV NS5B protein was involved in the template specificity of the enzyme. Site-directed mutagenesis of and prediction of the secondary structure of 3 terminal sequence of the template indicated that the cytidines at 3 terminus and the correct secondary structure of the template were essential to initiation of RNA synthesis by RdRp. Oxidation of the hydroxyl groups of the RNA template revealed that both the de novo initiation mechanism and the template-priming mechanism preference might be employed by the CSFV RdRp.     

Analysis of mutant NS5B proteins encoded by isolates from chimpanzees chronically infected following clonal HCV RNA inoculation

We hypothesized that mutations in the HCV NS5B polymerase, which occur during infection, may affect RNA-dependent RNA polymerase (RdRp) activity. NS5B proteins corresponding to a genotype 1a infectious clone and mutants identified in chimpanzees following inoculation with the clone were expressed and purified and their in vitro RdRp activity was compared to a NS5B genotype 1b control. A Gln-65-to-His mutation increased RdRp activity by 1.8-fold as compared to the infectious clone. Moreover, this NS5B1a protein had RdRp activity similar to the NS5B1b control. Three NS5B proteins representing mutations found in another animal had no in vitro RdRp activity. All mutations were maintained in the majority circulating virus for at least 216 weeks. The results demonstrate that some in vivo mutations of NS5B directly enhance in vitro RdRp activity. In addition, they suggest that the in vitro RdRp activity of NS5B may not always reflect in vivo activity within replication complexes.     

Mutations in NS5B polymerase of hepatitis C virus: impacts on in vitro enzymatic activity and viral RNA replication in the subgenomic replicon cell culture

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) essential for virus replication. Several consensus sequence motifs have been identified in NS5B, some of which have been shown to be critical for its enzymatic activity. A unique beta-hairpin structure located between amino acids 443 and 454 in the thumb subdomain has also been shown to play an important role in ensuring terminal initiation of RNA synthesis in vitro. However, the importance of these sequence and structural elements in viral RNA replication in infected cells has not been established, mainly due to the lack of a reliable cell culture system for HCV. In this study, we investigated the effect of several single amino acid substitutions and beta-hairpin truncations in NS5B on viral RNA replication by using the subgenomic replicon cell culture system. A strong correlation between in vitro polymerase activity and viral RNA replication was observed with most of the substitutions. Interestingly, truncations of the beta-hairpin (by four and eight amino acid residues, respectively), which did not reduce the in vitro enzymatic activity, completely abolished the ability of the replicon RNA to replicate in Huh-7 cells, demonstrating its essential role in viral RNA replication. Furthermore, a conservative substitution in motif D, from an arginine residue (AMTR(345)), which is conserved among all HCV isolates, to a lysine residue, resulted in significant improvements in both transient RNA replication and colony formation efficiencies. This result also correlates with a previous observation that the enzymatic activity of NS5B increased by about 50% when the same NS5B substitution was introduced (V. Lohmann, F. Korner, U. Herian, and R. Bartenschlager, J. Virol. 1997, 71, 8416-8428).     

Identification and characterization of RNA-dependent RNA polymerase activity in recombinant Japanese encephalitis virus NS5 protein

Summary The complete nonstructural NS5 gene of Japanese encephalitis virus (JEV) was amplified and cloned into an expression vector. The NS5 protein was expressed in Escherichia coli and purified by His-tag based affinity chromatography. This recombinant NS5 protein exhibited RNA-dependent RNA polymerase (RdRp) activity in vitro in the absence of other viral or cellular factors. The RNA polymerase activity was dependent on divalent cations, and Mn²⁺ was found to be 20 times more effective than Mg²⁺ in coordinating the catalytic reaction of RdRp, while Ca²⁺ inhibited enzyme activity. The optimal reaction conditions for the in vitro RdRp reaction were established. Characterization of the RdRp reaction products demonstrated that the JEV NS5 protein can initiate RNA synthesis through a de novo initiation mechanism in our in vitro reaction system. Comparing the efficiency of different RNA templates, we found that JEV NS5 protein was more efficient in using negative-strand RNA templates, indicating that the JEV NS5 protein is involved in regulating the ratio of positive- to negative-strand RNA. Four amino acid sequence motifs crucial for RdRp activity were also identified using site-directed mutagenesis analysis. All substitutions of the conserved residues within these motifs led to a complete inactivation or severe loss of enzyme activity.     

Classical swine fever virus NS5A regulates viral RNA replication through binding to NS5B and 3'UTR

In this report, classical swine fever virus (CSFV) NS5A inhibit viral RNA replication when its concentration reached and surpassed the level of NS5B. Three amino acid fragments of CSFV NS5A, 137-172, 224-268 and 390-414 individually were shown to be essential to NS5B binding. The former two fragments were independently necessary for regulation of viral RNA replication and correlated with NS5B and 3′UTR binding activity. We also found that amino acids W143, V145, P227, T246, P257, K399, T401, E406 and L413 of CSFV NS5A were essential to NS5B binding activity. Furthermore, these amino acids were shown to be necessary for viral RNA replication and infection and conserved in NS5A proteins of CSFV, BDV, BVDV and HCV. These results indicated that NS5A may regulate viral RNA replication by binding to NS5B and 3′UTR. NS5A can still regulate viral RNA synthesis through binding to 3′UTR when binding to NS5B is not available.     

日本血吸虫病患者血清对噬菌体随机7肽库的免疫筛选

目的 从噬菌体随机多肽文库中,筛选出能与慢性血吸虫病患者血清特异性结合的短肽分子。方法 采用慢性血吸虫病患者血清球蛋白作为配基,免疫筛选以融合蛋白形式在丝状噬菌体Ｍ１３外壳蛋白Ⅲ表达的随机７肽文库。按吸附－洗脱－扩增的淘筛过程,经３轮免疫淘筛后,随机挑取菌体克隆用ＥＬＩＳＡ检测其牧场 划性,并用斑点ＥＬＩＳＡ分析其诊断血吸虫病的价值。结果 经３轮免疫淘筛后,特异性结合的噬菌体富集增加近１００倍,有     

大肠杆菌脂多糖2630模拟位的研究

目的 :利用纯化的抗大肠杆菌L2 6 30多抗筛选噬菌体环七肽库 ,以获得可模拟脂多糖 (LPS)表位的短肽克隆。方法 :以亲和层析纯化的抗大肠杆菌L2 6 30多克隆抗体为靶分子 ,筛选噬菌体随机环七肽库 ,用双夹心ELISA和竞争抑制ELISA鉴定阳性噬菌体克隆的抗原性。结果 :对噬菌体环肽库进行 3轮筛选后 ,随机挑选 2 0个克隆 ,经鉴定其中 12个可与抗L2 6 30抗体结合 ,即为阳性克隆 ;其中有 5个阳性噬菌体克隆表现出与抗鼠伤寒沙门氏菌LPS多抗结合的活性 ,提示这 5个噬菌体展示的短肽具有模拟大肠杆菌LPS及鼠伤寒沙门氏菌LPS共同表位的性质。经DNA序列分析显示 ,其中 8个克隆的氨基酸序列具有X DGLL XX或X EDGLL X保守序列 ,其余 4个克隆的序列均不相同。结论 :筛选获得的噬菌体环七肽克隆具有模拟大肠杆菌LPS表位的活性 ,为大肠杆菌L2 6 30多表位模拟短肽。其中 5个阳性噬菌体克隆短肽具有模拟大肠杆菌LPS及鼠伤寒沙门氏菌LPS共同表位的活性     

Characterisation of interaction between NS3 and NS5B protein of classical swine fever virus by deletion of terminal sequences of NS5B

The NS3-NS5B interaction of classical swine fever virus (CSFV) is important for viral replication. For characterisation of the interaction between the NS3 and NS5B, a series of NS5B mutants with deletion of N-, C-terminal amino acids and quadruple alanine substitution mutations were produced. GST pull-down assays and immunoprecipitation analyses showed that NS5B and some NS5B mutants have NS3 binding activity. Further experimental data indicated that CSFV NS5B might contain two NS3 binding sites, one covering amino acids 63-99 located at the N-terminal end, another covering amino acids 611-642 at the C-terminal end. Assays for RNA-dependent RNA polymerase (RdRp) activity revealed that CSFV NS3 is able to enhance the RdRp activity of NS5B and some NS5B mutants in vitro. The enhancement might be obtained by NS3 binding to the two terminal sequences of NS5B, which could be attractive targets for drug development against CSFV.     

In vitro selection of RNA aptamers that bind the RNA-dependent RNA polymerase of hepatitis C virus: A possible role of GC-rich RNA motifs in NS5B binding

We employed SELEX (systematic evolution of ligands by exponential enrichment) and identified high affinity RNA aptamers to the hepatitis C virus NS5B RNA-dependent RNA polymerase (RdRp). GC-rich stretches were identified in many of the aptamers. Deletion of the 5′-end single-stranded GC-stretch (CGGG) of the highest binding RNA impaired the binding and the inhibitory activity of the RNA to NS5B RdRp. The majority of the mutants with a single base substitution on the CGGG motif exhibited weaker binding to NS5B. Interestingly, the CGGG motif is present on the stem structure of the NS5B coding RNA (5BSL3.2), which is considered to be an important cis-acting replication element. The 5BSL3.2 RNA showed substantial binding to NS5B, while a point mutation on the CGGG motif reduced the binding of RNA to NS5B. These results suggest a GC-stretch to be the RNA element recognized by NS5B.     

Mutational analysis of the GDD sequence motif of classical swine fever virus RNA-dependent RNA polymerases

To define the function of the GDD motif of the RNA-dependent RNA polymerase (RdRp) of classical swine fever virus (CSFV), single amino acid substitutions were introduced into the CSFV NS5B. All substitutions within the GDD motif were detrimental to the polymerase activity, the binding activity and the terminal nucleotidyl transferase activity of the NS5B protein. It was also found that the wild-type NS5B had higher RdRp activity with Mg(+2) than with Mn(+2) whereas some mutants worked better with Mn(+2) than with Mg(+2), suggesting that substitutions within the GDD motif modified the enzyme cation preferences and the GDD sequence of CSFV NS5B might be involved in polymerase-metal interaction. Therefore, the GDD amino acid sequence is important for the function of CSFV RdRp.     

丙型肝炎病毒N′-核心蛋白RNA结合区的定位

目的 对丙型肝炎病毒核心蛋白的RNA结合区进行详细定位，为制备缺失RNA结合活性突变体做准备。方法 构建GST融合丙型肝炎病毒核心蛋白不同区域片段表达质粒，在大肠杆菌中表达相应蛋白并用glutathione sepharose 4B进行纯化，利用二次电泳法检测RNA的结合活性，利用时相差电泳法比较不同区域RNA结合活性的强弱。结果 核心蛋白第1－132位氨基酸（AA）之间的片段得到了良好的表达和纯化，表达的蛋白经Western blot得到确认。二次电泳后的放射自显影结果显示，第1－10AA、19－45AA、80－132AA片段无RNA结合活性，而含有10－16AA，45－80AA的片段可结合单链和双链RNA。1－29AA与38－90AA2个片段对RNA的结合强度基本相同。结论 丙型肝炎核心蛋白中存在2个RNA结合区，分别定位于10－16AA及45－80AA。2个结合区都具有较强的RNA结合活性。制备RNA结合活性缺陷突变体须同时对2个结合区进行突变。