丙型肝炎病毒C区基因的克隆与分析

通过RT－PCR从1份来自甘肃武威地区献血员HCV阳性血清顺克隆到573bp的HCV核心基因全片段,用T－A克隆载体法将该片段接入克隆载体pUC19中并测序,结果表明武威地区分离株与Ⅰ型株HCV－Ⅰ和Ⅱ型株HCV－HeBei在该基因区段的核苷酸／氨基酸序列同源性分别为89.6%／95.4%、97.3%／97.4%,属于Ⅱ型株。     

Replication and infectivity of a novel genotype 1b hepatitis C virus clone

Hepatitis C virus infection is a major public health problem because of an estimated 170 million carriers worldwide. Genotype 1b is the major subtype of HCV in many countries and is resistant to interferon therapy. Study of the viral life cycle is important for understanding the mechanisms of interferon resistance of genotype 1b HCV strains. For such studies, genotype 1b HCV strains that can replicate and produce infectious virus particles in cultured cells are required. In the present study, we isolated HCV cDNA, which we named the NC1 strain, from a patient with acute severe hepatitis. Subgenomic replicon experiments revealed that several mutations enhanced the colony-formation efficiency of the NC1 replicon. The full-length NC1 genome with these adaptive mutations could replicate in cultured cells and produce infectious virus particles. The density gradient profile and morphology of the secreted virus particles were similar to those reported for the JFH-1 virus. Further introduction of a combination of mutations of the NS3 and NS5a regions into the NC1 mutants further enhanced secreted core protein levels and infectious virus titers in the culture medium of HCV-RNA-transfected cells. However, the virus infection efficiency was not sufficient for autonomous virus propagation in cultured cells. In conclusion, we established a novel cell culture-adapted genotype 1b HCV strain, termed NC1, which can produce infectious virus when the viral RNA is transfected into cells. This system provides an important opportunity for studying the life cycle of the genotype 1b HCV.     

Human immunodeficiency virus-1 Rev protein activates hepatitis C virus gene expression by directly targeting the HCV 5'-untranslated region

Coinfection with human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) accelerates hepatitis C disease progression; however, the mechanism underlying this effect is unknown. Here, we investigated the role of HIV-1 in HCV gene expression and the mechanism involved in this regulation. We discovered that HIV-1 Rev protein activates HCV gene expression. We further revealed that Rev binds to the internal loop of the HCV 5′-untranslated region (5′-UTR) to stimulate HCV IRES-mediated translation.     

Development of recombinant hepatitis C virus with NS5A from strains of genotypes 1 and 2

Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) plays multiple and diverse roles in the viral lifecycle, and is currently recognized as a novel target for anti-viral therapy. To establish an HCV cell culture system with NS5A of various strains, recombinant viruses were generated by replacing NS5A of strain JFH-1 with those of strains of genotypes 1 (H77; 1a and Con1; 1b) and 2 (J6CF; 2a and MA; 2b). All these recombinant viruses were capable of replication and infectious virus production. The replacement of JFH-1 NS5A with those of genotype 1 strains resulted in similar or slightly reduced virus production, whereas replacement with those of genotype 2 strains enhanced virus production as compared with JFH-1 wild-type. A single cycle virus production assay with a CD81-negative cell line revealed that the efficient virus production elicited by replacement with genotype 2 strains depended on enhanced viral assembly, and that substitutions in the C-terminus of NS5A were responsible for this phenotype. Pulse-chase assays revealed that these substitutions in the C-terminus of NS5A were possibly associated with accelerated cleavage kinetics at the NS5A–NS5B site. Using this cell culture system with NS5A-substituted recombinant viruses, the anti-viral effects of an NS5A inhibitor were then examined. A 300- to 1000-fold difference in susceptibility to the inhibitor was found between strains of genotypes 1 and 2. This system will facilitate not only a better understanding of strain-specific roles of NS5A in the HCV lifecycle, but also enable the evaluation of genotype and strain dependency of NS5A inhibitors.     

Codon usage of human hepatitis C virus clearance genes in relation to its expression

Hepatitis C virus (HCV) infection is among the leading causes of hepatocellular carcinoma and liver cirrhosis globally, with a high economic burden. The disease progression is well established, but less is known about the spontaneous HCV infection clearance. This study tries to establish the relationship between codon biasness and expression of HCV clearance candidate genes in normal and HCV infected liver tissues. A total of 112 coding sequences comprising 151 679 codons were subjected to the computation of codon indices, namely relative synonymous codon usage, an effective number of codon (Nc), frequency of optimal codon, codon adaptation index, codon bias index, and base compositions. Codon indices report of GC3s, GC12, hydropathicity, and aromaticity implicates both mutational and translational selection in the candidate gene set. This was further correlated with the differentially expressed genes among the selected genes using BioGPS. A significant correlation is observed between the gene expression of normal liver and cancerous liver tissues with codon bias (Nc). Gene expression is also correlated with relative codon bias values, indicating that CCL5, APOA2, CD28, IFITM1, and TNFSF4 genes have higher expression. These results are quite encouraging in selecting the high responsive genes in HCV clearance. However, there could be additional genes which could also orchestrate the clearance role with the above mentioned first line of defensive genes.     

Enhanced expression of matrix metalloproteinase-9 by hepatitis B virus infection in liver cells

The hepatitis B virus (HBV) is a major cause of human liver disease, including hepatocellular carcinoma (HCC). The prognosis for HCC is largely dependent on the clinicopathological characteristics regarding invasion and metastasis. Enhanced matrix metalloproteinase-9 (MMP-9) expression has been implicated as playing an important role in metastasis and invasion of HCC. However, the relationship between HBV infection and MMP-9 expression in HCC is currently poorly understood. We report here on a study of the levels of MMP-9 and MMP-2 expression in human fetal liver tissue, rat liver tissue, and Chang, HepG2, and Hep3B cells by gelatin zymography. Among these sources, Hep3B cells, which contain the integrated hepatitis B viral genome, continuously secrete the hepatitis B viral surface antigen, and express HBV genomic RNA, expressed high levels of proMMP-9, and a small amount of active MMP-9 was detected in Hep3B cells as assayed by zymography. We investigated the issue of whether HBV infection affects MMP-9 expression, which is known to play an important role in HCC invasion and metastasis. As a first step, human fetal hepatocyte (HFH) and HepG2 (HCC origin, HBV not detected) cells were subjected to infection with HBV, and the resulting infected cells successfully established are hereafter referred to as HFH-T2 and HepG2-HBV. The expression of MMP-9 was upregulated by the infected HBV in HFH-T2 and HepG2-HBV cells, as assayed by zymography, Northern blot, and Western blot analysis, and small amounts of active MMP-9 were detected in HFH-T2 and HepG2-HBV cells as assayed by zymography. The activation of the immature proMMP-9 to the mature MMP-9 could be induced by plasmin treatment. The activation of proMMP-9 was increased to a greater extent with plasmin treatment than without plasmin in HFH-T2 and HepG2-HBV cells but the addition of recombinant TIMP-1 inhibited the activation of proMMP-9. Finally, the addition of plasmin to the invasion assay using Matrigel resulted in an increase in invasiveness of HFH-T2 and HepG2-HBV cells, as well as MMP-9 activation, but the treatment with TIMP-1 inhibited the invasiveness of HFH-T2 and HepG2-HBV cells as well as MMP-9 activation. We conclude from these findings that HBV infection of hepatocytes and HepG2 cells affected the upregulation of MMP-9 expression and MMP-9 activation and, thus, increased the invasion potential by plasmin. To our knowledge, this is a first report showing that an HBV infection is linked to the upregulation of MMP-9 in HCC.     

丙型肝炎病毒C+E1区基因在原核细胞中的克隆与表达

将丙型肝炎病毒Ｃ＋Ｅ１区基因插入到原核高效表达载体ｐＢＶ２２１质粒中,构建了质粒ｐＢＶ２２１ＨＣＶ／Ｃ＋Ｅ１作为表达载体,然后,将含有该质粒的宿主大肠杆菌进行升温诱导表达ＨＣＶ／Ｃ＋Ｅ１区基因,并对表达产物进行了生物活性的检测。结果表明,插入到表达载体ｐＢＶ２２１中的ＨＣＶ／Ｃ＋Ｅ１基因片段能够得到有效的表达,表达产物主要为非融合蛋白形式存在于细胞中,同时这种Ｃ区和Ｅ１区连接共表达的产物保持了良好的抗原活性     

High expression of APOBEC3G in patients infected with hepatitis C virus

APOBEC3G (an apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; also known as CEM15), a member of the APOBEC family, which possesses cytidine deaminase activity that causes C/G to T/A transition mutations in virus genomes such as human immunodeficiency virus 1 and hepatitis B virus, is reported to play an important role in host-defense mechanisms. However, APOBEC3G expression in patients infected with chronic hepatitis C virus (HCV), of which there are currently more than 170 million worldwide, has not yet been well studied. We investigated this issue herein, and demonstrated an increased expression of APOBEC3G in both hepatocytes and lymphocytes of chronic hepatitis patients infected with HCV. Transfection of the NS5A gene, but not any other non-structural protein genes of HCV tested, to the hepatocellular carcinoma cell line enhanced APOBEC3G expression. Incubation of the cells with interferon also resulted in the augmentation. These results may provide new insight into the pathogenesis of chronic HCV infection.     

Sequential biphasic changes in claudin1 and claudin4 expression are correlated to colorectal cancer progression and liver metastasis

Terminal progression of colorectal cancer (CRC) culminates in liver metastasis. To identify genes that are involved in the metastatic phenotype, cDNA microarrays were used to analyse mRNA expression profiles of colorectal carcinoma (CC)531 rat colon adenocarcinoma cells for changes related to their homing into the liver. Briefly, CC531 cells were intraportally implanted into the liver of Wag-Rij rats and re-isolated after 3, 6, 9, 14 and 21 days. Compared to control CC531 cells, claudin1 and claudin4 were among the ≥8-fold initially down-regulated genes. The co-culture of tumour cells with isolated rat hepatocytes and Kupffer cells did not induce down-regulation of either claudin1 or 4. When the environment effective on circulating tumour cells was simulated by cell culture conditions favouring their adhesion, only claudin4 showed augmented expression. Knockdown of claudin1 and claudin4 mediated by small interfering RNA caused significantly increased migration and decreased clonogenic growth of tumour cells (P < 0.05), but had no effect on their proliferation. These experimental results were paralleled by increased claudin1 and claudin4 expression in human CRC samples in Union for International Cancer Control (UICC) stages I-III, as evaluated by real-time PCR. Increased claudin4 levels were correlated with significantly reduced overall survival (log-rank test, P= 0.018). Further, significantly (P < 0.05) reduced expression of claudin1 and claudin4 was observed in stage IV and liver metastasis by immunohistochemistry. In conclusion, sequential biphasic changes in claudin1 and claudin4 expression occur during the homing of rat CC531 CRC cells to the liver. This modulation is reflected by significant changes in claudin expression in human primary and metastatic CRC.     

Bim-mediated apoptosis and PD-1/PD-L1 pathway impair reactivity of PD1(+)/CD127(-) HCV-specific CD8(+) cells targeting the virus in chronic hepatitis C virus infection

PD-1 molecule promotes anergy and IL-7 receptor (CD127) induces an anti-apoptotic effect on T cells. Correlation between PD-1/CD127 phenotype and hepatitis C virus (HCV)-specific CD8⁺ cell reactivity in resolved infection (RI) after treatment and persistent HCV-infection (PI) was analysed. Directly ex vivo, PD-1 and CD127 expression on HCV-specific CD8⁺ cells displayed a positive and negative correlation, respectively with viraemia. Proliferation after stimulation on PD-1⁻/CD127⁺ cells from RI cases was preserved, while it was impaired on PD-1⁺/CD127⁻ cells from PI patients. PD1⁺/CD127⁺ population was observed in PI, and these maintained expansion ability but they did not target the virus. Frequency of PI cases with HCV-specific CD8⁺ cell proliferation increased after anti-PD-L1 and anti-apoptotic treatment. Bim expression on HCV-specific CD8⁺ cells from PI patients was enhanced. In conclusion, during chronic HCV infection non-reactive HCV-specific CD8⁺ cells targeting the virus are PD-1⁺/CD127⁻/Bim⁺ and, blocking apoptosis and PD-1/PD-L1 pathway on them enhances in vitro reactivity.     

Influence of increased CD4 cell counts on the genetic variability of hepatitis C virus in patients co-infected with human immunodeficiency virus I.

In order to study the effect of increased CD4 cell counts on the biology of hepatitis C virus (HCV), we analyzed the genetic variability of HCV generated over 8 y in eight human immunodeficiency virus-1 (HIV-1) and HCV co-infected patients. This was a retrospective study in which HIV patients were selected who had profound immune impairment evident over four years and were co-infected with HCV genotype 1 and who then went on highly active antiretroviral therapy (HAART). These patients achieved different degrees of immune reconstitution, measured as increased CD4 cell counts during a 4- to 8-y period, following initiation of HAART. HCV genetic variability was determined by measuring the genetic diversity (Hamming distance, HD), and complexity (number of viral variants) in plasma samples collected at yearly intervals just before and after the initiation of HAART. The parameters were assessed by molecular cloning and sequencing of a 575-bp fragment including the HCV envelope 1 and envelope 2 genes (E1/E2), containing the hypervariable region 1 (HVR1). significantly increased HVR1 genetic diversity was observed in analyzed samples where the patients' CD4 cell counts were > or =100 compared with CD4 cell counts <100. A significant increase in genetic diversity in HVR1 was detected in co-infected patients whose CD4 cell counts increased from <100 to >400 over a period of more than 4 y of HAART therapy. This was in contrast to a minimal increase in HCV genetic diversity of HVR1 occurring in patients whose CD4 cell counts failed to rise much over 200 over 7 y of follow up. Insertion and deletion of HCV genomic fragments in the E1/E2 region was documented in one patient who developed fulminant hepatitis C.     

丙肝病毒核心基因靶向性M1GS 核酶的体外抗病毒活性

[目的]丙型肝炎病毒(Hepatitis C Virus,HCV)是引起病毒性肝炎的重要病原.目前临床HCV感染多采用干扰素联合病毒唑进行治疗,但其应答率低且感染易反复,探索新型抗HCV治疗策略与药物具有紧迫的现实意义.[方法]基于大肠埃希菌来源的核糖核酸酶P(RNase P),针对HCV核心基因的序列,设计一小段与之互补的外部引导序列(Guide Sequence,GS),通过PCR将其共价连接至大肠埃希菌RNase P催化性亚基(M1 RNA)的3'末端,从而构建一种靶向性的核酶——M1GS.[结果]构建的M1 GS-HCV/C52核酶在体外可对靶RNA片段产生特异性切割;在HCV感染的Huh7.5.1细胞,该人工核酶也能够显著抑制HCV核心基因的表达,并使培养上清中HCV RNA的拷贝数减少约1500倍.[结论]本研究构建的M1GS-HCV/C52核酶具有显著的体外抗病毒活性,从而为HCV的治疗研究提供了一条潜在途径.     

Hepatitis C virus nonstructural protein NS3 binds to Sm-D1, a small nuclear ribonucleoprotein associated with autoimmune disease

Hepatitis C virus (HCV) causes a persistent infection, chronic hepatitis, and leads to hepatocellular carcinoma. Nonstructural protein 3 (NS3) of HCV possesses protease, nucleoside triphosphatase, and helicase activities. Using the yeast two hybrid assay, we identified Sm-D1, a host protein that binds to NS3. Sm-D1 is a component of small nuclear ribonucleoprotein (snRNP) complexes which are associated with autoimmune disease. Sm-D1 has Gly-Arg (GR) repeats at the C terminus, which contains dimethylarginine modified by post-translational modification and may constitute an immunoreactive determinant. Deletion mutants revealed that the C-terminal region of Sm-D1 containing the GR repeats was the binding region for NS3, and the expression feature of Sm-D1 was affected by co-expression of NS3. Immunostaining assay demonstrated that NS3 was also present in the nucleus of cells overexpressing Sm-D1, although it was usually found in cytoplasm. The localization of NS3 could change following interaction with Sm-D1 and affect the function of Sm-D1.     

Infection of human hepatocyte chimeric mouse with genetically engineered hepatitis C virus and its susceptibility to interferon

We developed a reverse genetics system of hepatitis C virus (HCV) genotypes 1a and 2a using infectious clones and human hepatocyte chimeric mice. We inoculated cell culture-produced genotype 2a (JFH-1) HCV intravenously. We also injected genotype 1a CV-H77C clone RNA intrahepatically. Mice inoculated with HCV by both procedures developed measurable and transmissible viremia. Interferon (IFN) alpha treatment resulted in greater reduction of genotype 2a HCV levels than genotype 1a, as seen in clinical practice. Genetically engineered HCV infection system should be useful for analysis of the mechanisms of resistance of HCV to IFN and other drugs.