丙肝病毒NS5A蛋白对NS5B的RdRP活性影响的研究

丙型肝炎病毒 (Hepatitis C virus,HCV )非结构蛋白 (Nonstructural,NS) 5 A在 HCV基因组复制中的作用目前尚不清楚。本文研究 His- NS5 A对 NS5 B的 RNA依赖性 RNA酶 (Rd RP)活性的影响 ,以了解 NS5 A在HCV RNA复制中的作用。采用变性 -复性方法 ,纯化大肠杆菌表达的重组组氨酸 NS5 A融合蛋白。 GST结合洗脱实验 (GST pull- down assay)研究 NS5 A和 NS5 B是否结合。以不同的摩尔浓度比 ,将纯化的 NS5 B和 NS5 A蛋白混合 ,检测 NS5 A对 NS5 B的 Rd RP活性的影响。获得高得率的纯化 His- NS5 A蛋白。重组 NS5 A蛋白可在体外与NS5 B结合并抑制后者的 Rd RP活性。本研究报道了纯化重组 His- NS5 A蛋白的变性 -复性方法 ,结果显示纯化的重组 NS5 A在体外可与 NS5 B相互结合 ,并明显抑制 NS5 B Rd RP活性。提示了 HCV NS5 A在病毒复制中的可能作用。     

Functional Interactions between Conserved Motifs of the Hepatitis C Virus RNA Helicase Protein NS3

The hepatitis C virus NS3 gene encodes a RNA helicase with several sequence motifs conserved among the members of the DExH box protein family. The contributions of the sequence motifs to enzyme activity were assessed in this study by substitution of alanine for the Lys in the ATP binding motif GxGK (referred to as K1236A mutation), or for the Asp in the DExH motif (D1316A), or for the Arg in the middle of the QRxGRxGR motif known for RNA binding (R1490A). Histidine-tagged recombinant proteins of Mr 54,000 were expressed in Escherichia coli and purified by chromatography on nickel agarose. All three mutants were severely defective in ATPase and RNA helicase activities, but loss of the ATPase activity was not dependent on polynucleotide cofactors. With the exception of R1490A mutant, a stable complex was formed between dsRNA substrates and recombinant proteins, indicating that the arginine-rich motif is required for efficient RNA binding. Complex formation was not affected by omission of ATP or substitution by a non-hydrolyzable analog AMP-PCP, suggesting that neither binding nor hydrolysis of ATP is required for RNA binding. Moreover, the K1236A mutant which was defective in binding ATP exhibited an unusually strong affinity for RNA duplex. These results suggest that the conserved motifs cooperatively constitute a large functional domain rather than act as individual domains with strictly independent functions, and that alteration of one motif affects functions of other motifs in a mutually interactive fashion.     

Antigenic Epitopes of the Hepatitis A Virus Polyprotein

Forty-two antigenic domains were identified across the hepatitis A virus (HAV) polyprotein by using a set of 237 overlapping 20-mer synthetic peptides spanning the entire HAV polyprotein and a panel of serum samples from acutely HAV-infected patients. The term "antigenic domain" is used in this study to define a protein region spanned with consecutive overlapping immunoreactive peptides. Nineteen antigenic domains were found within the structural proteins, and 22 were found within the nonstructural proteins, with 1 domain spanning the junction of VP1 and P2A proteins. Five of these domains were considered immunodominant, as judged by both the breadth and the strength of their immunoreactivity. One domain is located within the VP2 protein at position 57-90 aa. A second domain, located at position 767-842 aa, contains the C-terminal part of the VP1 protein and the entire P2A protein. A third domain, located at position 1403-1456 aa, comprises the C-terminal part of the P2C protein and the N-terminal half of the P3A protein. The fourth domain, located at position 1500-1519 aa, includes almost the entire P3B, and the last domain, located at position 1719-1764 aa, contains the C-terminal region of the P3C protein and the N-terminal region of the P3D protein. It is interesting to note that four of the five most immunoreactive domains are derived from small HAV proteins and/or encompass protein cleavage sites separating different HAV proteins. The HAV-specific immunoreactivity of each antigenically reactive peptide was confirmed by using seven HAV seroconversion panels. Collectively, these data demonstrate that HAV structural and nonstructural proteins contain antigenic epitopes that can be efficiently modeled with short synthetic peptides.     

Hepatitis C Virus Genotyping Using an Oligonucleotide Microarray Based on the NS5B Sequence

The genotype of the hepatitis C virus (HCV) is essential for determining treatment duration in clinical practice and for epidemiological and clinical studies. Currently, few genotyping assays that determine the HCV subtype are available. This report describes a microarray-based molecular technique for identifying the HCV genotype and subtype. It uses low-density hydrogel-based biochips containing genotype- and subtype-specific oligonucleotides based on the sequences of the NS5B region of the HCV genome. The biochip contains 120 oligonucleotides that identify genotypes 1 to 6 and 36 (1a, 1b, 1c, 1d, 1e, 2a, 2b, 2c, 2d, 2i, 2j, 2k, 2l, 2m, 3a, 3b, 3k, 4a, 4c, 4d, 4f, 4h, 4i, 4k, 4n, 4o, 4p, 4r, 4t, 5a, 6a, 6b, 6d, 6g, 6h, and 6k) subtypes. The procedure included amplification of a 380-nucleotide (nt) fragment of NS5B and its hybridization on the biochip. Tests on 345 HCV-positive samples showed that the assay agreed with NS5B sequencing 100% for the genotype and 99.7% for the subtype. The hybridization on the microarray and the NS5B sequence were in 100% agreement for identifying the most common subtypes, 1a, 1b, 4a, 4d, and 3a. This approach is a promising tool for HCV genotyping, especially for implementing the new anti-HCV drugs that require accurate identification of clinically relevant subtypes.The hepatitis C virus (HCV) is a leading cause of chronic liver disease and increased risk of cirrhosis and hepatocellular carcinoma (51). More than 170 million people are infected with HCV worldwide (42). This enveloped, single-stranded positive-sense RNA virus is a member of the Flaviviridae family. The RNA genome contains a single large open reading frame composed of over 9,000 nucleotides (nt) encoding structural and nonstructural proteins (5). One of these proteins is an RNA-dependent RNA polymerase encoded by the so-called NS5B region. This error-prone enzyme lacks proofreading activity, which makes it responsible for the great genetic variability of HCV. Sequencing studies of HCV strains have identified 6 genotypes and more than 70 subtypes (43, 45).The HCV genotype is considered to be the major baseline predictor of a sustained virological response (SVR) to antiviral therapy. Patients infected with HCV genotypes 2 and 3 are more sensitive to combination therapy with interferon and ribavirin than are those infected with genotype 1 (8, 11, 21). The available data on HCV genotype 4 suggest that its sensitivity to HCV treatment lies somewhere between those of genotypes 1 and 2/3 (17). The sensitivity of genotypes 5 and 6 could be similar to that of genotype 2 or 3 (1, 9, 19). The HCV subtype has recently been implicated as a potential predictor of SVR. One study of 597 difficult-to-treat patients found that subtypes 1b, 4a, and 4d were independently associated with SVR (16). The virological response to new anti-HCV agents could also be influenced by the HCV subtype (31, 42).Several methods has been proposed for HCV genotyping (50), including commercially available techniques based on real-time PCR: the HCV genotyping analyte-specific reagent (ASR) assay (Abbott Molecular Inc., Des Plaines, IL) (23), semiautomated sequencing (the TruGene HCV 5′NC genotyping kit; Bayer HealthCare, Berkeley, CA) (10), and automated reverse hybridization (the Inno-LiPA HCV II assay; Innogenetics, Ghent, Belgium) (46, 49). Most HCV genotyping methods are based on analysis of the 5′ noncoding (NC) region of the HCV genome because the 5′ NC region is regularly amplified for HCV molecular diagnosis and quantification of the viral load. However, this highly conserved region is not suitable for accurately discriminating between subtypes and can lead to genotyping or subtyping errors (2, 3, 15, 39, 43). Hence, alternative genomic regions have been proposed for genotyping HCV, including the core fragment (35, 49) and the NS5B region (39). Sequencing and phylogenetic analysis of the NS5B region are presently considered to be the gold standard for HCV genotyping since they accurately identify the subtype and can be used to establish an epidemiological picture of circulating virus strains (27, 30, 39, 47). However, this method includes steps of purification of the amplified product, sequencing, and phylogenetic analysis that require the skill of laboratory personnel, a factor that can be a limitation to the wide use of the technique in routine clinical laboratories.Therefore, an assay was developed that involves hybridization on an oligonucleotide microarray for identifying HCV genotypes and subtypes. It uses a low-density hydrogel-based microarray (biochip) that has been successfully used in many fields of molecular diagnostics (26, 28, 37). The microarray contains genotype- and subtype-specific oligonucleotides based on the corresponding sequences of the NS5B region. This report compares this approach to accurately identifying HCV genotype and subtype with direct NS5B sequencing.     

Confirmation of Nosocomial Transmission of Hepatitis C Virus by Phylogenetic Analysis of the NS5-B Region

Four hepatitis C virus transmission chains at three dialysis units were disclosed by limited sequencing; three of these were disclosed by analysis of the NS5-B region of the genome. Dialysis on the same shift as that during which infected patients were dialyzed was the common factor for seven patients in two chains. Two nurses exposed to needle sticks and their sources of infection constituted two other chains. The strains of three chains belonged to subtype 1a and formed clusters with an intrachain variability of 0 to 6 nucleotides compared to 8 to 37 nucleotides for unrelated strains within this subtype. The clusters were supported by bootstrap values ranging from 89 to 100%.     

Antigenic Domains of the Open Reading Frame 2-Encoded Protein of Hepatitis E Virus

The antigenic composition of the hepatitis E virus (HEV) protein encoded by open reading frame 2 (ORF2) was determined by using synthetic peptides. Three sets of overlapping 18-, 25-, and 30-mer peptides, with each set spanning the entire ORF2 protein of the HEV Burma strain, were synthesized. All synthetic peptides were tested by enzyme immunoassay against a panel of 32 anti-HEV-positive serum specimens obtained from acutely HEV-infected persons. Six antigenic domains within the ORF2 protein were identified. Domains 1 and 6 located at the N and C termini of the ORF2 protein, respectively, contain strong immunoglobulin G (IgG) and IgM antigenic epitopes that can be efficiently modeled with peptides of different sizes. In contrast, antigenic epitopes identified within the two central domains (3 and 4) were modeled more efficiently with 30-mer peptides than with either 18- or 25-mers. Domain 2 located at amino acids (aa) 143 to 222 was modeled best with 25-mer peptides. A few 30-mer synthetic peptides derived from domain 5 identified at aa 490 to 579 demonstrated strong IgM antigenic reactivity. Several 30-mer synthetic peptides derived from domains 1, 4, and 6 immunoreacted with IgG or IgM with more than 70% of anti-HEV-positive serum specimens. Thus, the results of this study demonstrate the existence of six diagnostically relevant antigenic domains within the HEV ORF2 protein.     

Generation of Immune Responses against Hepatitis C Virus by Dendritic Cells Containing NS5 Protein-Coated Microparticles

Dendritic cells (DCs) internalize and process antigens as well as activate cellular immune responses. The aim of this study was to determine the capacity of DCs that contain antigen-coated magnetic beads to induce immunity against the nonstructural hepatitis C virus (HCV) antigen 5 (NS5). Splenocytes derived from Fms-like tyrosine kinase receptor 3 (Flt3) ligand-pretreated BALB/c mice were incubated with magnetic beads coated with HCV NS5, lipopolysaccharide (LPS), and/or anti-CD40; purified; and used for immunization. Cellular immunity was measured using cytotoxic T-lymphocyte (CTL) and T-cell proliferation assays, intracellular cytokine staining, and a syngeneic tumor challenge using NS5-expressing SP2/0 myeloma cells in vivo. Splenocytes isolated from animals vaccinated with DCs containing beads coated with NS5, LPS, and anti-CD40 secreted elevated levels of interleukin-2 (IL-2) and gamma interferon in the presence of NS5. The numbers of CD4⁺, IL-2-producing cells were increased >5-fold in the group immunized with DCs containing beads coated with NS5, LPS, and anti-CD40, paralleled by an enhanced splenocyte proliferative response. Immunization promoted antigen-specific CTL activity threefold compared to the level for control mice and significantly reduced the growth of NS5-expressing tumor cells in vivo. Thus, strategies that employ NS5-coated beads induce cellular immune responses in mice, which correlate well with the natural immune responses that occur in individuals who resolve HCV.An estimated 170 million individuals (3% of the world''s population) are infected with hepatitis C virus (HCV), leading to cirrhosis, end-stage liver disease, and hepatocellular carcinoma. The current standard-of-care therapy for chronically infected HCV patients is the combined administration of pegylated alpha interferon (IFN-α) and ribavirin (28). A sustained response is seen in approximately 50 to 60% of individuals (33). Treatment is long term (6 to 12 months), costly, and associated with substantial toxicity (42). Clearly, more-effective regimens are needed (10).Clinical studies of the adaptive host immune response to acute HCV infection suggest a rationale for immunization approaches. Clearance of HCV is associated with early, multispecific, strong CD8⁺ T-cell immunity that is matched by vigorous and sustained CD4⁺ T-cell proliferation in response to multiple recombinant structural and nonstructural viral proteins (13, 18, 47). Activated T cells secrete proinflammatory cytokines (TH1 type), such as IFN-γ, coinciding with large reductions in viral load during acute infection (47). HCV infections that are successfully controlled result in durable memory populations (44). This observation is supported by a substantially lower rate of HCV persistence in reexposed humans with a history of acute resolving HCV (29). Rechallenge experiments with chimpanzees showed that antibody-mediated depletion of CD4⁺ T cells resulted in HCV persistence, which is in contrast to a marked reduction in duration and peak of viremia in nontreated animals (4, 20). The importance of CD4⁺ T cells is further emphasized by the loss of immune protection against reexposure to HCV and correlated with low CD4⁺ T-cell counts in intravenous drug users who had recovered from HCV but subsequently acquired human immunodeficiency virus infection (29). Whether recovery from acute HCV coincides ultimately with virus eradication is still a matter of debate (34). The disappearance of HCV-specific antibodies in some individuals 10 to 20 years after viral clearance indicates that a subgroup of patients achieves complete virus elimination (44).Persistent HCV infection can be attributed to a number of viral evasion strategies. First, the rapid spread of HCV in a host outpaces the immune response by several weeks (39, 46). Second, HCV is a strong inducer of type I IFN but appears to render hepatocytes resistant to antiviral activity (45, 46). Third, NK cell function can be inhibited by cross-linking tetraspanin CD81 on the cell surface with the major envelope protein of HCV (HCV E2), thus blocking NK cell activation, proliferation, and cytokine production (12). Finally, persistent HCV infections correlate with the permanent loss of HCV-specific T-cell proliferation during acute HCV infection, the functional exhaustion of an initially vigorous response, and the inability of effector T cells to migrate into the infected liver (10, 13, 18, 36, 39, 46). Insufficient CD4⁺ T-cell activity appears to be a key event leading or contributing to chronic HCV. Failure to sustain the CD4⁺ helper response renders virus-specific CD8⁺ T cells inadequate (e.g., loss of cytotoxicity and IFN-γ production), thus contributing to persistent viremia (39).Recapitulating successful immune responses and addressing HCV-specific defects in immunity are mandatory in efforts to improve current treatment options. The purported involvement of dendritic cells (DCs) in the impaired immune responses observed in patients with persistent HCV infection makes DCs a principal target for immunomodulatory approaches. Access to a sufficient quantity of mature DCs is a critical issue since only mature DCs are capable of targeting the antigen and inducing cellular immunity rather than tolerance. Previously, we outlined a novel method of generating large numbers of DCs in vivo (17). These, in turn, are enriched in vitro by phagocytosis of magnetic beads and separation in a magnetic field. Here we further demonstrate that, by immunizing mice with DCs that contain beads coated with the nonstructural HCV antigen NS5 and compounds known to induce DC maturation, significant levels of cellular immunity are elicited. The key elements of this approach reside in the combined enrichment, maturation, and antigen targeting of DCs in a single step and the generation of immune responses of the type known to promote viral clearance in humans.     

Hepatitis C virus core, NS3, NS5A, NS5B proteins induce apoptosis in mature dendritic cells

Although reasons for hepatitis C virus (HCV) persistence are still unknown, specific cellular immune responses appear to influence the pathogenesis and outcome of the infection. Apoptosis of cells infected by viruses may appear suicidal to the viruses that induce programmed cell death of its host. However, apoptosis has been suggested to be a response to virus infection as a mean of facilitating virus dissemination. Annexin V-propidium iodide staining and DNA fragmentation, were used to show that expression of the core, NS3, NS5A, or NS5B protein induces apoptosis in mature dendritic cells. In addition, immunoblotting was used to demonstrate that expression level of p21waf1/cip1 protein decreased in cells expressing one of these HCV proteins. No expression of p53 could be detected and expression of Akt was independent of HCV proteins expression. These results suggest that the effect of these HCV proteins on HCV associated pathogenesis may be linked (at least partially) to its ability to modulate apoptosis pathways in mature dendritic cells.     

Evidence That Hepatitis C Virus Resistance to Interferon Is Mediated through Repression of the PKR Protein Kinase by the Nonstructural 5A Protein

Hepatitis C virus (HCV) is the major cause of non-A non-B hepatitis and a leading cause of liver dysfunction worldwide. While the current therapy for chronic HCV infection is parenteral administration of type 1 interferon (IFN), only a fraction of HCV-infected individuals completely respond to treatment. Previous studies have correlated the IFN sensitivity of strain HCV-1b with mutations within a discrete region of the viral nonstructural 5A protein (NS5A), termed the interferon sensitivity determining region (ISDR), suggesting that NS5A may contribute to the IFN-resistant phenotype of HCV. To determine the importance of HCV NS5A and the NS5A ISDR in mediating HCV IFN resistance, we tested whether the NS5A protein could regulate the IFN-induced protein kinase, PKR, a mediator of IFN-induced antiviral resistance and a target of viral and cellular inhibitors. Using multiple approaches, including biochemical, transfection, and yeast genetics analyses, we can now report that NS5A represses PKR through a direct interaction with the protein kinase catalytic domain and that both PKR repression and interaction requires the ISDR. Thus, inactivation of PKR may be one mechanism by which HCV avoids the antiviral effects of IFN. Finally, the inhibition of the PKR protein kinase by NS5A is the first described function for this HCV protein.     

丙型肝炎病毒F蛋白对p21基因的调节作用

目的研究丙型肝炎病毒（HCV）F蛋白对细胞p21基因转录、表达的影响。方法PCR扩增HCV1b来源的F基因，克隆至pcDNA3．1真核表达载体。F基因质粒转染HepG2细胞，48h后抽提细胞总RNA和总蛋白，实时定量PCR及Westem blot检测p21基因表达变化。结果HCVF蛋白在HepG2细胞内瞬时表达，相对于空载体对照（目标基因表达量为1），HCVF基因质粒转染细胞的p21转录水平为3．2，蛋白表达水平为1．4。结论HCVF蛋白增加p21转录和表达，其生物学效应值得进一步研究。     

Correlation between Mutations in the Interferon Sensitivity-Determining Region of NS5A Protein and Viral Load of Hepatitis C Virus Subtypes 1b, 1c, and 2a

In the present study, we analyzed the possible relationship between interferon (IFN) sensitivity-determining region (ISDR) sequence variation of various hepatitis C virus (HCV) subtypes and serum HCV titers in Indonesian patients without IFN treatment. The viremia titers (mean +/- standard deviation) of HCV subtype 1b (HCV-1b) isolates with low (three or fewer) and high (four or more) numbers of ISDR mutations were 5.4 +/- 0.6 and 4.2 +/- 0.9 log(10) RNA copies/ml, respectively, with the difference between the two groups being statistically significant (P < 0.01). Similarly, the viremia titers of HCV-1c isolates with low and high numbers of ISDR mutations were 5.3 +/- 0.6 and <3.0 +/- 0.0 log(10) RNA copies/ml, respectively, with the difference between the two groups being statistically significant (P < 0.01). Also, the virus titers of HCV-2a isolates with low and high numbers of ISDR mutations were 4.3 +/- 0.7 and 3.5 +/- 0.4 log(10) RNA copies/ml, respectively, with the difference between the two groups being statistically significant (P < 0.01). Thus, our results demonstrated that virus load in Indonesian patients infected with HCV-1b, HCV-1c, or HCV-2a correlated inversely with the number of mutations in the ISDR sequence, implying the possibility that the ISDR sequence plays an important role in determining the levels of HCV viremia.     

Hepatitis C virus NS5A protein protects against TNF-alpha mediated apoptotic cell death

Hepatitis C virus (HCV) often causes a prolonged and persistent infection which may lead to hepatocellular carcinoma. We have previously reported that the nonstructural 5A (NS5A) protein of HCV promotes cell growth [Ghosh, A.K., Steele, R., Meyer, K., Ray, R., Ray, R.B., 1999. Hepatitis C virus NS5A protein modulates cell cycle regulatory genes and promotes cell growth. J. Gen. Virol. 80, 1179-1183]. In this study, we investigated the role of HCV NS5A (genotype 1a, strain H) in TNF-alpha induced apoptotic cell death. HepG2 cells expressing NS5A exhibited an inhibitory role in relation to TNF-alpha mediated apoptotic cell death. The NS5A protein blocked the activation of caspase-3 and inhibited proteolytic cleavage of the death substrate poly (ADP-ribose) polymerase in TNF-alpha induced cells. Together, these results suggest that HCV NS5A protein protects against TNF-alpha mediated apoptotic cell death.     

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.     

High-Resolution Hepatitis C Virus Subtyping Using NS5B Deep Sequencing and Phylogeny,an Alternative to Current Methods

Hepatitis C virus (HCV) is classified into seven major genotypes and 67 subtypes. Recent studies have shown that in HCV genotype 1-infected patients, response rates to regimens containing direct-acting antivirals (DAAs) are subtype dependent. Currently available genotyping methods have limited subtyping accuracy. We have evaluated the performance of a deep-sequencing-based HCV subtyping assay, developed for the 454/GS-Junior platform, in comparison with those of two commercial assays (Versant HCV genotype 2.0 and Abbott Real-time HCV Genotype II) and using direct NS5B sequencing as a gold standard (direct sequencing), in 114 clinical specimens previously tested by first-generation hybridization assay (82 genotype 1 and 32 with uninterpretable results). Phylogenetic analysis of deep-sequencing reads matched subtype 1 calling by population Sanger sequencing (69% 1b, 31% 1a) in 81 specimens and identified a mixed-subtype infection (1b/3a/1a) in one sample. Similarly, among the 32 previously indeterminate specimens, identical genotype and subtype results were obtained by direct and deep sequencing in all but four samples with dual infection. In contrast, both Versant HCV Genotype 2.0 and Abbott Real-time HCV Genotype II failed subtype 1 calling in 13 (16%) samples each and were unable to identify the HCV genotype and/or subtype in more than half of the non-genotype 1 samples. We concluded that deep sequencing is more efficient for HCV subtyping than currently available methods and allows qualitative identification of mixed infections and may be more helpful with respect to informing treatment strategies with new DAA-containing regimens across all HCV subtypes.     

丙型肝炎病毒NS3蛋白单克隆杂交瘤细胞株的建立及初步鉴定

应用国产基因工程表达的丙型肝炎病毒（ＨＣＶ）ＮＳ３区抗原免疫小鼠，然后取其脾细胞与小鼠骨髓瘤细胞系ＳＰ２／０融合，筛选出４株稳定分泌抗ＨＣＶＮＳ３区蛋白单克隆抗体（ＭｃＡｂ）的杂交瘤细胞株，分别命名为２Ｂ６，２Ｆ３，３Ｄ８，３Ｄ９，经初步研究表明这４株单抗与ＮＳ３抗原具有良好的反应性，与ＨＣＶ核心区多肽及乙型肝炎病毒表面抗原和ｅ抗原均无反应。抑制实验表明这４株抗体分别针对ＮＳ３抗原分子上的２个不同的抗原决定簇。     

Hepatitis C virus-host interactions: the NS5A protein and the interferon/chemokine systems.

The interactions that occur between viral proteins and host factors, such as cellular proteins and signal transduction machinery, have a significant influence on the replication, persistence, and pathogenesis of all viruses. This is exemplified by hepatitis C virus (HCV), which infects an estimated 3% of the world's population and is a significant cause of liver disease. HCV-host interactions also affect the outcome of interferon (IFN) antiviral therapy, which is effective only in certain patients. In this review, we focus on the HCV nonstructural 5A (NS5A) protein, a model for diverse virus-host interactions, and highlight the interaction of viruses, including HCV, with the chemokine system.     

Expression and Membrane Association of Hepatitis C Virus Envelope 1 Protein

The expression of hepatitis C virus (HCV) E1 protein is toxic for Escherichia coli cells. For this reason, we have cloned the E1 gene in the pET3a vector and analyzed the inducible expression of the protein in two strains of E. coli characterised by a different level of reduction of basal synthesis. The results indicated that synthesis of E1 was supported only by the BL21(DE3)pLysS strain which provides a tightest control of protein expression before the induction. The BL21(DE3)pLysS cells were then used for the expression of E1 gene, varying at its carboxy terminus in order to retain (E1, aa 192–383) or delete (E1t, aa 192–340) a C-terminal hydrophobic region that may be involved in membrane association. Following cell fractionation, E1 protein was found associated with the membrane fraction. By contrast, the truncated mutant E1t, was identified in the soluble phase suggesting a direct role for the C-terminal domain in E1 membrane association.