一个协议，用于分析丙型肝炎病毒复制
Summary
Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.
Abstract
丙型肝炎病毒（HCV）影响世界人口的3％，并导致严重的肝脏疾病，包括慢性肝炎，肝硬化和肝细胞癌。丙型肝炎病毒是属于黄病毒科的包膜RNA病毒。目前的治疗是不充分有效的，并导致不利的副作用。没有丙型肝炎病毒疫苗可用。因此，需要开发一种疫苗和更好的治疗持续的努力。的HCV细胞培养体系是研究HCV生长的不同阶段，包括病毒进入，基因组复制，包装和出口的关键。在当前的程序提出，我们用野生型intragenotype 2a中的嵌合病毒，FNX-HCV和重组FNX-的Rluc病毒携带的Renilla荧光素酶报道基因来研究病毒的复制。一个人肝癌细胞株（Huh-7型）用于体外转录转染丙型肝炎病毒基因组RNA的。无细胞培养物上清液，蛋白裂解液和叔OTAL核糖核酸收获的不同时间点转染后，以评估HCV生长。丙型肝炎病毒基因组的复制状态是由定量RT-PCR和可视化HCV的存在双链RNA进行评估。丙型肝炎病毒蛋白的表达，用特异性HCV NS3和NS5A蛋白的抗体，Western blot和免疫荧光试验验证。感染性颗粒释放到培养上清液中和病毒滴度的HCV RNA转染的细胞进行测定。荧光素酶检测被用于评估记者丙型肝炎病毒的复制水平和传染性。总之，我们提出的各种病毒学检测表征丙型肝炎病毒复制周期的不同阶段。
Introduction
丙型肝炎病毒（HCV）导致肝硬化和肝癌。它影响1.7亿人在全球拥有35万人，每年死于1-3。 HCV是正链RNA病毒为9.6 kb的基因组大小。 HCV基因组被翻译成的〜3000氨基酸残基被蛋白水解由各种细胞和病毒蛋白酶裂解成10多肽的单个蛋白。 HCV是原型病毒属中的肝炎病毒，属于黄病毒科 4。曝光后，建立了丙型肝炎病毒慢性感染者中80％的个体。这种感染大多是无症状的，及时的诊断可以允许治疗性干预，以防止肝恶化。目前的治疗是不理想的，没有疫苗可用5,6。
丙型肝炎的病因在1989年7年首次描述。学习丙型肝炎病毒复制是丙型肝炎的疫苗和治疗研究的重要，但它一直由缺少一种有效的病毒培养体系的长期阻碍。丙型肝炎病毒的分子克隆被证明是在感染后肝内接种8只黑猩猩。随后，HCV亚基因组复制子进行了说明，其中允许解剖病毒基因组的复制阶段中的细胞培养系统9,10。一个基因型2a丙型肝炎病毒的发现隔离JFH-1（日本暴发性肝炎-1），能够感染细胞培养中打开丙型肝炎病毒复制的研究11-13新途径。基因型2a株JFH-1基于跨内和基因型嵌合病毒基因型1丙型肝炎病毒感染的基础培养体系可作为以及14-18。
我们已成功地使用JFH-1株和HCV intragenotype 2a中的嵌合病毒，得到蛋白质结构域和顺式作用RNA元件19,20的高分辨率官能剖析图。根据这一点，我们在这里描述的有效培养系统经常使用的，允许研究HCV复制周期和宿主 – 病原体相互作用的不同阶段。我们目前的病毒学检测，以评估病毒基因组的复制和intragenotype 2A丙型肝炎病毒和海肾萤光素酶报告基于 丙型肝炎病毒新发感染。
Protocol
Representative Results
Discussion
此图描述了用于分析丙型肝炎病毒复制循环的方法。丙型肝炎病毒是一种人类病原体和规定的生物安全议定书将必须严格遵守。感染性HCV细胞培养系统之前已经11-13,16,17说明。有下列图示的协议，当我们实现几个关键点。首先，它是非常重要，以有完整的全长病毒基因组RNA质量好于下游的研究。输入质粒携带的病毒cDNA要进行线性化处理，并仔细减弱。的绿豆核酸酶来生硬的Xba I位悬…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank F. Chisari for providing Huh-7.5.1 cell line. We would like to thank Justine Ho for editing the manuscript. This work was supported by Cedars-Sinai Medical Center Institutional Programmatic Research Award and National Center for Advancing Translational Sciences, Grant UL1TR000124 to V.A.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Dulbecco’s modified Eagle’s medium (DMEM) | Fisher Scientific | 10-017-CV | |
| Non essential amino acid | Fisher Scientific | MT25025CI | |
| HEPES | Life Technologies | 15630080 | |
| Glutamax | Life Technologies | 35050061 | |
| Opti-MEM Reduced Serum Medium,no Phenol Red | Life Technologies | 11058-021 | |
| Huh-7.5.1 | The Scripps Research Institute | The cell line was kindly provided by Dr. Francis Chisari to Dr. Arumugaswami under executed MTA between The Scripps Research Institute and Cedars-Sinai Medical Center | |
| Plasmids (pFNX-HCV, pFNX-HCV Pol null, pFNX-Rluc, and pFNX-Rluc Pol null) | Cedars-Sinai Medical Center | The HCV plasmids were synthesized by Dr. Arumugaswami using overlapping oligo-nucleotides. | |
| XbaI | New England Biolabs Inc. | R0145S | |
| Mung Bean Nuclease | New England Biolabs Inc. | M0250S | |
| T7 RiboMAX Express Large Scale RNA Production System | Promega | P1320 | |
| Rneasy Mini Kit | Qiagen | 74104 | |
| Nanodrop 2000 | Thermo Scientific | Nanodrop 2000 | |
| Electroporation Cuvette (4 mm) | Bioexpress | E-5010-4 | |
| Gene Pulser Xcell Total System | Bio-Rad | 165-2660 | |
| mouse monoclonal anti-dsRNA antibody J2 | English & Scientific Consulting Kft. | 10010200 | |
| Goat anti-rabbit IgG Alexa Fluor 488 | Life Technologies | A11008 | |
| Goat anti-rabbit IgG Alexa Fluor 594 | Life Technologies | A11020 | |
| PVDF membrane package | Bio-Rad | 162-0263 | |
| Blotting Grade Blocker Non Fat Dry Milk | Bio-Rad | 170-6404XTU | |
| Tween-20 | Bio-Rad | 170-6531XTU | |
| Anti-Hepatitis C Virus NS3 antibody [8 G-2] | Abcam | ab65407 | |
| Anti-Hepatitis C Virus NS3 antibody [H23] | Abcam | ab13830 | |
| Goat anti-mouse IgG conjugated with horseradish peroxidase (HRP) | Jackson ImmunoResearch Laboratories Inc. | 115-035-003 | |
| Amersham ECL Prime Western Blotting Detection Reagents | GE Healthcare Life Sciences | RPN2236 | |
| SUPERSCRIPT III RT | Life Technologies | 18080085 | |
| SYBR QPCR SUPERMIX W/ROX | Life Technologies | 11744500 | |
| ViiA 7 real-time PCR system | Life Technologies | NA | |
| Renilla Luciferase Assay System kit | Promega | E2810 | |
| RNase-Free DNase | Promega | M6101 | |
| GloMax-Multi Detection System (Luminometer) | Promega |
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