Robust full-length hepatitis C virus genotype 2a and 2b infectious cultures using mutations identified by a systematic approach applicable to patient strains

Hepatitis C virus (HCV) infection is a leading cause of chronic liver diseases worldwide, but treatment options are limited. Basic HCV research required for vaccine and drug development has been hampered by inability to culture patient isolates, and to date only the JFH1 (genotype 2a) recombinant replicates spontaneously in hepatoma cells and releases infectious virus. A JFH1 chimera with the 5' end through NS2 from another genotype 2a strain, J6, had enhanced infectivity. However, the full-length J6 clone (J6CF), which we previously found to be fully functional in vivo, was replication incompetent in vitro. Through a systematic approach of culturing J6 with minimal JFH1 sequences, we identified three mutations in NS3, NS4A, and NS5B that permitted full-length J6 propagation and adaptation with infectivity titers comparable to JFH1-based systems. The most efficient recombinant, J6cc, had six adaptive mutations and did not accumulate additional changes following viral passage. We demonstrated that HCV NS3/NS4A protease-, NS5A- and NS5B polymerase-directed drugs respectively inhibited full-length J6 infection dose dependently. Importantly, the three J6-derived mutations enabled culture adaptation of the genetically divergent isolate J8 (genotype 2b), which differed from the J6 nucleotide sequence by 24%. The most efficient recombinant, J8cc, had nine adaptive mutations and was genetically stable after viral passage. The availability of these robust JFH1-independent genotype 2a and 2b culture systems represents an important advance, and the approach used might permit culture development of other isolates, with implications for improved individualized treatments of HCV patients and for development of broadly efficient vaccines.     

Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras

Chronic liver disease caused by infection with hepatitis C virus (HCV) is an important global health problem that currently affects 170 million people. A major impediment in HCV research and drug development has been the lack of culture systems supporting virus production. This obstacle was recently overcome by using JFH1-based full-length genomes that allow production of viruses infectious both in vitro and in vivo. Although this improvement was important, because of the restriction to the JFH1 isolate and a single chimera consisting of J6CF and JFH1-derived sequences, broadly based comparative studies between different HCV strains were not possible. Therefore, in this study we created a series of further chimeric genomes allowing production of infectious genotype (GT) 1a, 1b, 2a, and 3a particles. With the exception of the GT3a/JFH1 chimera, efficient virus production was obtained when the genome fragments were fused via a site located right after the first transmembrane domain of NS2. The most efficient construct is a GT2a/2a chimera consisting of J6CF- and JFH1-derived sequences connected via this junction. This hybrid, designated Jc1, yielded infectious titers 100- to 1,000-fold higher than the parental isolate and all other chimeras, suggesting that determinants within the structural proteins govern kinetic and efficiency of virus assembly and release. Finally, we describe an E1-specific antiserum capable of neutralizing infectivity of all HCV chimeras.     

Biliverdin inhibits hepatitis C virus nonstructural 3/4A protease activity: mechanism for the antiviral effects of heme oxygenase?

Induction of heme oxygenase-1 (HO-1) inhibits hepatitis C virus (HCV) replication. Of the products of the reaction catalyzed by HO-1, iron has been shown to inhibit HCV ribonucleic acid (RNA) polymerase, but little is known about the antiviral activity of biliverdin (BV). Herein, we report that BV inhibits viral replication and viral protein expression in a dose-dependent manner in replicons and cells harboring the infectious J6/JFH construct. Using the SensoLyte 620 HCV Protease Assay with a wide wavelength excitation/emission (591 nm/622 nm) fluorescence energy transfer peptide, we found that both recombinant and endogenous nonstructural 3/4A (NS3/4A) protease from replicon microsomes are potently inhibited by BV. Of the tetrapyrroles tested, BV was the strongest inhibitor of NS3/4A activity, with a median inhibitory concentration (IC(50)) of 9 μM, similar to that of the commercial inhibitor, AnaSpec (Fremont, CA) #25346 (IC(50) 5 μM). Lineweaver-Burk plots indicated mixed competitive and noncompetitive inhibition of the protease by BV. In contrast, the effects of bilirubin (BR) on HCV replication and NS3/4A were much less potent. Because BV is rapidly converted to BR by biliverdin reductase (BVR) intracellularly, the effect of BVR knockdown on BV antiviral activity was assessed. After greater than 80% silencing of BVR, inhibition of viral replication by BV was enhanced. BV also increased the antiviral activity of α-interferon in replicons. Conclusion: BV is a potent inhibitor of HCV NS3/4A protease, which likely contributes to the antiviral activity of HO-1. These findings suggest that BV or its derivatives may be useful in future drug therapies targeting the NS3/4A protease.     

Identification of specific regions in hepatitis C virus core,NS2 and NS5A that genetically interact with p7 and co‐ordinate infectious virus production

The p7 protein of hepatitis C virus (HCV) is a small, integral membrane protein that plays a critical role in virus replication. Recently, we reported two intergenotypic JFH1 chimeric viruses encoding the partial or full‐length p7 protein of the HCV‐A strain of genotype 1b (GT1b; Virology; 2007; 360:134). In this study, we determined the consensus sequences of the entire polyprotein coding regions of the wild‐type JFH1 and the revertant chimeric viruses and identified predominant amino acid substitutions in core (K74M), NS2 (T23N, H99P) and NS5A (D251G). Forward genetic analysis demonstrated that all single mutations restored the infectivity of the defective chimeric genomes suggesting that the infectious virus production involves the association of p7 with specific regions in core, NS2 and NS5A. In addition, it was demonstrated that the NS2 T23N facilitated the generation of infectious intergenotypic chimeric virus encoding p7 from GT6 of HCV.     

Studies on virus kinetics using infectious fluorescence‐tagged hepatitis C virus cell culture

Aim: Studies of the complete hepatitis C virus (HCV) life cycle have become possible with the development of a HCV‐JFH1 cell culture system. Methods: In this study, we constructed two fluorescence protein‐tagged recombinant JFH1 virus clones, JFH1‐EYFP and JFH1‐AsRed, as well as two corresponding clones with adaptive mutations, JFH1‐EYFP mutant and JFH1‐AsRed mutant, that and were as effective as JFH1 in producing infectious virus particles, and investigated their viral infection life cycles. Results: After infection of the fluorescence‐tagged mutant viruses, infected cells increased exponentially. In cells, EYFP or AsRed and NS5A were expressed as a fusion protein and co‐localized in core proteins. The rate of the cell–cell spread was dependent on the cell densities with a maximum of 10^2.5/day. Treatment of cells with interferon or a protease inhibitor suppressed expansion of virus‐positive cells. Conclusion: Taken together, these results indicate that fluorescence‐tagged HCV is a useful tool to study virus infection life cycles and to assist in the search for novel antiviral compounds.     

Integrity of the NS5A (amino acid 2209 to 2248) region in hepatitis C virus 1b patients non‐responsive to interferon therapy

Abstract: Background/Aims: In hepatitis C virus‐1b, it has been suggested that an amino acid stretch (aa 2209–2248) of the carboxy terminal half of the non‐structural 5A (NS5A) region participates in the response to interferon treatment. We tested the hypothesis that absence of mutations in the NS5A (aa 2209–2248) sequence is required for interferon resistance. We also investigated the importance of different HCV‐1b isolates in interferon response in France. Methods: We determined the NS5A sequences of 70 patients with chronic hepatitis C before IFN therapy and then compared them with HCV‐J prototype sequence. The isolates were determined by NS5B sequencing, the “gold standard” method for genotyping and subtyping. Pre‐therapeutic viral load was also measured. Results: No sustained virological response was observed in the patients without amino acid substitutions in the NS5A (aa 2209–2248) sequence, and in the patients with HCV‐J isolates. Viral load was significantly higher in the patients with no amino acid substitutions in the NS5A (aa 2209–2248) sequence. Conclusions: In HCV‐1b infected patients, an HCV‐J strain with no amino acid substitution in the NS5A (aa 2209–2248) region indicates a poor prognosis for response to IFN therapy. The low interferon response rate in HCV‐1b infection in Europe is probably not due to a difference between isolates.     

An infectious and selectable full-length replicon system with hepatitis C virus JFH-1 strain

Aim: The hepatitis C virus (HCV) strain JFH‐1 was cloned from a patient with fulminant hepatitis. A JFH‐1 subgenomic replicon and full‐length JFH‐1 RNA efficiently replicate in cultured cells. In this study, an infectious, selectable HCV replicon containing full‐length JFH‐1 cDNA was constructed. Methods: The full‐genome replicon was constructed using the neomycin‐resistant gene, EMCV IRES and wild‐type JFH‐1 cDNA. Huh7 cells were transfected with RNA synthesized in vitro, and then cultured with G418. Independent colonies were cloned to establish cell lines that replicate the full‐length HCV replicon. Results: HCV RNA replication was detected in each isolated cell line. HCV proteins and HCV RNA were secreted into culture medium, and exhibited identical density profiles. Interestingly, culture supernatants of the replicon cells were infectious for naïve Huh7 cells. Long‐term culture did not affect replication of replicon RNA in the replicon cells, but it reduced core protein secretion and infectivity of culture supernatant. Culture supernatant obtained after serial passage of replicon virus was infectious for Huh7 cells. Conclusions: Selectable infection was established using HCV replicon containing full‐length genotype 2a JFH‐1 cDNA. This system might be useful for HCV research.     

基于2a基因型丙型肝炎病毒自身启动子的单顺反子复制子的构建和功能测定

目的 构建丙型肝炎病毒(HCV)单顺反子复制子,研究其在Huh7.5和Huh7.1细胞中的复制功能,为研究HCV复制规律和抗病毒药物的筛选建立模型. 方法 用Quick change点突变方法删除pJ6JFH 1B1aRL质粒上的Core-E1-E2-p7-NS2片段(约3090 bp),得到△pJ6JFH1B1aRL,然后测序,选择序列正确的克隆,用AgeⅠ和AvrⅡ酶切回收目的片段约2280bp,同时用AgeⅠ和AvrⅡ酶切pSGRJFH1和其突变体质粒,回收载体片段.将目的片段和载体片段连接,构建由HCV-IRES启动的单顺反子复制子pSGRm-JFH IblaRL以及其变异突变体pSGRm-JFH 1b1aRLGND,用其RNA转染Huh7.5和Huh7.1细胞,研究复制子在细胞中的复制情况.结果 经过Quick change和多步克隆方法成功构建HCV-IRES启动的单顺反子复制子,复制子RNA转染Huh7.5和Huh7.1细胞72h后复制达高峰,96h复制开始下降,而对照的突变体从24h至96h均没有复制.结论 成功构建了HCV-IRES单顺反子复制子,转染Huh7.5和Huh7.1细胞后在不同时间均有复制.     

Highly efficient full-length hepatitis C virus genotype 1 (strain TN) infectious culture system

Chronic infection with hepatitis C virus (HCV) is an important cause of end stage liver disease worldwide. In the United States, most HCV-related disease is associated with genotype 1 infection, which remains difficult to treat. Drug and vaccine development was hampered by inability to culture patient isolates representing HCV genotypes 1–7 and subtypes; only a recombinant 2a genome (strain JFH1) spontaneously replicated in vitro. Recently, we identified three mutations F1464L/A1672S/D2979G (LSG) in the nonstructural (NS) proteins, essential for development of full-length HCV 2a (J6) and 2b (J8) culture systems in Huh7.5 cells. Here, we developed a highly efficient genotype 1a (strain TN) full-length culture system. We initially found that the LSG substitutions conferred viability to an intergenotypic recombinant composed of TN 5′ untranslated region (5′UTR)-NS5A and JFH1 NS5B-3′UTR; recovered viruses acquired two adaptive mutations located in NS3 and NS4B. Introduction of these changes into a replication-deficient TN full-length genome, harboring LSG, permitted efficient HCV production. Additional identified NS4B and NS5B mutations fully adapted the TN full-length virus. Thus, a TN genome with 8 changes (designated TN cell-culture derived, TNcc) replicated efficiently and released infectious particles of ∼5 log₁₀ focus-forming units per mL; passaged TNcc did not require additional changes. IFN-α and directly acting antivirals targeting the HCV protease, NS5A, and NS5B, each inhibited full-length TN infection dose-dependently. Given the unique importance of genotype 1 for pathogenesis, this infectious 1a culture system represents an important advance in HCV research. The approach used and the mutations identified might permit culture development for other HCV isolates, thus facilitating vaccine development and personalized treatment.Hepatitis C virus (HCV) chronically infects an estimated 130–170 million people worldwide. The infection increases the risk of developing liver cirrhosis and liver cancer and results in more than 350,000 deaths annually. No HCV vaccine is available. Current standard treatment is based on IFN-α/ribavirin, which, however, has low efficacy against the most prevalent HCV variants (1). Incorporation of directly acting antivirals (DAAs) in treatment regimens improves sustained viral response rate, but a favorable outcome is challenged by fast emergence of drug resistance and differential responses of the different HCV genotypes (2). Thus, HCV infection continues to be a huge health and economic burden to the world population, and improved in vitro experimental systems would be important to permit additional studies of new antivirals and associated resistance patterns.HCV is a small enveloped virus belonging to the genus Hepacivirus in the family Flaviviridae. The HCV genome is a positive-sense single-strand RNA (∼9.6 kb), consisting of a single ORF flanked by 5′ and 3′ untranslated regions (UTRs). The ORF encodes virus structural proteins (core, E1, and E2), p7, and six nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B (1). HCV isolates are classified into seven major variants (genotypes 1–7) and numerous subtypes (a, b, etc.) differing by ∼30% and ∼20%, respectively, in nucleotide and amino acid sequences (3). Genotype 1 is the most prevalent in the world. In the United States, Japan, China, and southern Europe over 70% of HCV patients are infected with genotype 1 (4–7). In northern Europe, genotype 1 accounts for ∼50% of HCV infections (8). Genotype 1 infection is more resistant to IFN-based treatment than infection with other genotypes (1).In vitro HCV culture systems are needed to study treatment regimens and associated viral resistance. However, drug and vaccine development have been greatly hampered by inability to culture patient isolates in vitro. Although a number of HCV full-length clones were shown to be infectious in chimpanzees (ref. 9 and cited references therein), only JFH1 (genotype 2a) spontaneously replicated in human hepatoma cells (Huh7 and its derivatives) and released infectious virus particles (10, 11). Efficient growth of JFH1 required culture adaptive mutations (12). Recently, we reported efficient J6cc (2a) and J8cc (2b) full-length culture systems (13); subsequently Date et al. reported on a full-length system for 2a strain JFH2 (14). A single full-length genotype 1a genome, H77-S, carrying mutations identified in the subgenomic replicon of the same strain, has been reported to release relatively small amounts of virus particles (15). The Con1 (1b) full-length culture system was reported, but a very low level of replication has limited its utility (16). Thus, efficient HCV full-length culture systems remained limited to genotype 2 isolates. The clinical importance of the marked genetic differences between HCV genotype isolates poses a critical need for development of robust full-length culture systems for HCV genotype 1 isolates.The unique replication capacity of JFH1 has permitted the development of JFH1-based HCV recombinants (17); we and others have reported different inter- and intragenotypic recombinants including core-NS2 (17–24), 5′UTR-NS2 (25), NS3 protease/NS4A (26, 27), NS5A (28), and core-NS3 protease plus NS4A-NS5A (29) of various genotypes. These JFH1-based culture systems have been used for genotype-specific studies of HCV genes (19, 20, 22, 25–28, 30), for testing of HCV DAAs (26–28) and neutralizing antibodies (19, 20, 22, 30), and for studying aspects of virus–host interaction (25, 31, 32). Through studies of J6 (2a) recombinants with the entire or partial NS5B and 3′UTR from JFH1, we recently identified adaptive mutations F1464L in NS3 and A1672S in NS4A, designated LS (13). Combination of LS with selected mutations in NS5B and the 3′UTR allowed replication of the full-length J6 genome, which led to the identification of an additional unique mutation D2979G in NS5B, designated G (13). The LSG substitutions permitted the development of robust HCV full-length culture systems for J6 and for the prototype genotype 2b J8 strain (13). In this study, we used LSG as a critical component and a unique approach to develop a highly efficient full-length genotype 1a (strain TN) infectious culture system, named TN cell-culture derived (TNcc). The TNcc replicated efficiently in culture, released viral particles of ∼10⁵ focus-forming units (FFU)/mL and did not require additional mutations after viral passage. We demonstrated that full-length TN responded dose dependently to IFN-α, as well as HCV DAAs already used in the clinic and being tested in clinical trials.     

Genotype differences in susceptibility and resistance development of hepatitis C virus to protease inhibitors telaprevir (VX-950) and danoprevir (ITMN-191)

Protease inhibitors (PIs) have proven to be effective adjuncts to interferon/ribavirin treatment of hepatitis C virus (HCV) infections. Little clinical or in vitro data exists, however, on their effectiveness for nontype 1 genotypes that predominate in Europe, the Middle East, Africa, and most of Asia. NS3 protease and NS4A genes from genotypes 1-6 were inserted into the JFH clone to generate replication-competent intergenotype chimeras. Susceptibility to PIs was determined by replication and infectivity assays. To study resistance development, chimeras were cultured in subinhibitory concentrations of PIs and mutations phenotypically characterized. Marked differences in susceptibility of different genotypes to danoprevir (ITMN-191) and telaprevir (VX-950) were observed. Genotypes 1, 4, and 6 showed median inhibitory concentration (IC(50) ) values of 2-3 nM, >100-fold lower than genotypes 2/3/5 (250-750 nM). Telaprevir susceptibilities varied over a 4-fold range, with genotypes 1 and 2 being most susceptible and genotypes 4 and 5 most resistant. Culture of genotypes 1-6 in PIs induced numerous mutations in the NS3 protease domain, highly variable between genotypes. Introduction of danoprevir and BILN 2061-induced mutations into the original clones by site-directed mutagenesis (n = 29) all conferred resistant phenotypes, with particularly large increases (1-2 log greater IC(50) values) in the initially susceptible genotypes 1/4/6. Most introduced mutations and showed little or no effect on replicative fitness. CONCLUSION: Major differences were found between genotypes in their susceptibility and resistance development to PIs. However, equal sensitivities of genotypes 1, 4, and 6 to danoprevir and a broader efficacy range of telaprevir between genotypes than initially conceptualized provide strong evidence that PIs might be effectively used beyond their genotype 1 target group.     

Influence of RNA titre and amino acid changes in the NS5A region of GB virus c/hepatitis G virus on the effectiveness of interferon therapy

BACKGROUND: A relationship between the pretreatment RNA titre of GB virus C/hepatitis G virus (GBV-C/HGV) and the effectiveness of interferon (IFN) therapy has been reported previously. However, the influence of changes in the amino acid sequence of the NS5A region of GBV-C/HGV on the effectiveness of IFN therapy has not been examined, although this influence has been explored in patients with chronic hepatitis caused by hepatitis C virus. We examined the relationship between changes in the amino-acid sequence of the NS5A region and the effectiveness of IFN therapy. METHODS: The subjects were 10 patients with chronic hepatitis C coinfected with GBV-C/HGV and treated with IFN. The pretreatment level of GBV-C/HGV-RNA (copies/mL) in their sera was measured by real-time detection polymerase chain reaction (PCR) assay. At 6 months after cessation of therapy, four of 10 patients had become negative for GBV-C/HGV-RNA (CR, complete response) and six patients were still positive for GBV-C/HGV-RNA (NR, non-response). We determined the nucleotide sequence of the NS5A region (amino acid residues 1865-2279; NS5A1865-2279) of pretreatment GBVC/HGV-RNA by direct sequencing. RESULTS: The pretreatment GBV-C/HGV-RNA level of CR patients (7.8 x 10(4) - 6.2 x 10(5), mean 3.30 x 10(5)) was significantly lower than that of NR patients (6.3 x 10(7) - 7.2 x 10(8), mean 3.55 x 10(8); P< 0.01). The number of amino acid substitutions in NS5A1865-2279 was five to seven (mean 5.8 +/- 1.0) in CR patients, and four to eight (mean 6.8 +/- 1.6) in NR patients, a difference that is not significant. Moreover, there were no amino acid substitutions or sites of substitution in NS5A1865-2279 that were specific to either group. CONCLUSIONS: The effectiveness of IFN therapy for GBV-C/HGV is strongly related to the pretreatment GBV-C/HGV-RNA level, but is not related to changes in NS5A1865-2279.     

Hepatitis C virus NS5A promotes insulin resistance through IRS-1 serine phosphorylation and increased gluconeogenesis

AIM: To investigate the mechanisms of insulin resistance in human hepatoma cells expressing hepatitis C virus(HCV) nonstructural protein 5A(NS5A).METHODS: The human hepatoma cell lines,Huh7 and Huh7.5,were infected with HCV or transientlytransfected with a vector expressing HCV NS5 A. The effect of HCV NS5 A on the status of the critical players involved in insulin signaling was analyzed using realtime quantitative polymerase chain reaction and Western blot assays. Data were analyzed using Graph Pad Prism version 5.0.RESULTS: To investigate the effect of insulin treatment on the players involved in insulin signaling pathway,we analyzed the status of insulin receptor substrate-1(IRS-1) phosphorylation in HCV infected cells or Huh7.5 cells transfected with an HCV NS5 A expression vector. Our results indicated that there was an increased phosphorylation of IRS-1(Ser307) in HCV infected or NS5 A transfected Huh7.5 cells compared to their respective controls. Furthermore,an increased phosphorylation of Akt(Ser473) was observed in HCV infected and NS5 A transfected cells compared to their mock infected cells. In contrast,we observed decreased phosphorylation of Akt Thr308 phosphorylation in HCV NS5 A transfected cells. These results suggest that Huh7.5 cells either infected with HCV or ectopically expressing HCV NS5 A alone have the potential to induce insulin resistance by the phosphorylation of IRS-1 at serine residue(Ser307) followed by decreased phosphorylation of Akt Thr308,Fox01 Ser256 and GSK3β Ser9,the downstream players of the insulin signalingpathway. Furthermore,increased expression of PECK and glucose-6-phosphatase,the molecules involved in gluconeogenesis,in HCV NS5 A transfected cells was observed.CONCLUSION: Taken together,our results suggest the role of HCV NS5 A in the induction of insulin resistance by modulating various cellular targets involved in the insulin signaling pathway.     

丙型肝炎NS5ATP6基因原核表达载体的构建和表达纯化及多克隆抗体的制备

目的构建丙型肝炎病毒NS5A蛋白反式激活蛋白6基因的原核表达载体并进行表达、鉴定。方法从已构建的pGBKT7-NS5ATP6质粒上切取NS5ATP6基因,再克隆入pET32a( )质粒,构建pET32a( )-NS5ATP6表达重组体。结果以pET32a( )-NS5ATP6重组体分别转化DH5α和Rosseta大肠埃希菌后,经IPTG诱导,pET32a( )-NS5ATP6表达出分子量为41KD左右的重组蛋白。免疫动物并经Westernblot检测证实其具有良好的抗原性。结论成功地构建了原核表达载体pET32a( )-NS5ATP6,诱导表达和纯化了NS5ATP6融合蛋白,并制备了该蛋白的多克隆抗体,为下一步该基因功能研究奠定了基础。     

丙型肝炎病毒非结构蛋白5A反式激活基因NS5ATP13的克隆化研究

目的 应用微矩阵(microauray)技术，结合生物信息学(bioinformatics)技术筛选并克隆丙型肝炎病毒(HCV)非结构蛋白5A(NS5A)的反式激活基因，阐明授性HCV感染、肝纤维化及肝细胞癌(HCC)的等相关疾病的发病机制。方法 根据HCV-H病毒株序列设计、合成序列特异性的引物。以含有全长HCV—H株cDNA的pBRTM-3011质粒DNA作为模板，进行多聚酶链反应(PCR)扩增，获得的HCVNS5A编码基因片段克隆到TA载体中进行核昔酸序列的测定，构建真核表达载体PcDNA3．1(-)-NS5A。以pcDNA3．1(-)-NS5A转染肝母细胞瘤细胞系HepG2，提取总RNA，逆转录为cDNA后进行表达谱基因芯片分析。应用分子生物学技术，结合生物信息学技术，克隆HCVKDA反式激活作用的新的靶基因。结果 构建了真核表达载体pcDNA3．1(-)-NS5A，经过限制性内切酶作图分析和核苔酸序列分析证实正确无误。以PcDMA3．1(-)-NS5A转染HepG2后提取总MA，逆转录后进行表达谱基因芯片技术分析。应用分子克隆技术结合生物信息学技术克隆NS5A反式激活的新型靶基因，命名为NS5ATP13，在GenBank中登录，登录号为D21262。Ns5ATP13基因的编码序列全长为2103个核苷酸(nt)，编码产物由700个氨基酸残基(aa)组成。结论 丙型肝炎病毒NS5A基因产物具有显著的反式激活作用。微短阵技术是分析基因表达谱变化的自动化和高通量技术。应用这些技术，发现了HCVKDA反式激活作用的新的靶基因，将为进一步研究HCVNS5A反式激活作用的分子生物学机制及HCV相关疾病发病机制奠定基础。     

Evolution of hepatitis C virus non-structural 5A gene in the progression of liver disease to hepatocellular carcinoma.

BACKGROUND: The interaction between the hepatitis C virus (HCV) non-structural 5A (NS5A) protein of HCV and the protein kinase R (PKR), which is an effector of the cellular antiviral response and has been defined as a tumour suppressor, may affect the control of protein synthesis and cell growth. AIM: We investigated the genetic evolution of the NS5A region in the NS5A PKR-binding domain (NS5A-PKRbd) of patients with HCV 1b-related cirrhosis who subsequently developed or not hepatocellular carcinoma (HCC). Patients and Methods: The quasispecies composition of NS5A-PKRbd was inferred by sequencing an average of 15 clones per sample in specimens obtained from 26 patients with cirrhosis who developed or not HCC during a follow-up of 5 years. RESULTS: At baseline, 13/17 patients with final HCC and six out of nine patients with cirrhosis who subsequently did not develop HCC harboured a wild-type (wt) strain master sequence. Over time, the prevalence of wt strain was higher in patients who developed HCC with respect to those who maintained the cirrhosis status (15/17 vs 4/9, respectively; P=0.0166). CONCLUSION: The maintenance of or evolution to the wt strain of the NS5A domain in cirrhotic patients with final HCC highlights the central role of NS5A protein in the viral life cycle and in the progression of liver disease.     

丙型肝炎病毒全长基因嵌合萤光素酶重组质粒转染细胞系的建立与初步应用

目的 构建能产生较高效价重组病毒的HCV细胞感染模型,为HCV致病机制的研究和抗病毒药物的筛选提供一个有效的体外细胞培养系统.方法 利用PCR技术在HCV NS5A C端插入海肾萤光素酶( Renilla Luciferase,Rluc)报告基因,并引入能提高HCV效价的突变,酶切鉴定重组基因序列构建成功后,转染入...