1 / 27

HCV 复制子的研究与应用

HCV 复制子的研究与应用. 第三军医大学西南医院感染病分院 全军感染病研究所 王 小 红. HCV 研究存在的问题. HCV 疫苗研究尚未获得突破进展 急需新型的抗 HCV 药物 研究主要障碍： 缺乏合适的细胞模型及小动物模型. HCV 细胞模型的发展. 1992 年 各种细胞培养系统. 1994 年 肝细胞系. 人 T 细胞系 ： MOLT-4Ma ， HPB-Ma ， H9 ， MT-2 人骨髓源淋巴样细胞系： CE ， TOFE 人 B 细胞系 ： Daudi. 原代黑猩猩和人肝细胞 WRL68 ， HepG2

matt
Download Presentation

HCV 复制子的研究与应用

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. HCV 复制子的研究与应用 第三军医大学西南医院感染病分院 全军感染病研究所 王 小 红

  2. HCV研究存在的问题 • HCV疫苗研究尚未获得突破进展 • 急需新型的抗HCV药物 研究主要障碍： 缺乏合适的细胞模型及小动物模型

  3. HCV 细胞模型的发展 1992年 各种细胞培养系统 1994年 肝细胞系 人T细胞系：MOLT-4Ma，HPB-Ma，H9 ，MT-2 人骨髓源淋巴样细胞系：CE，TOFE 人B细胞系：Daudi 原代黑猩猩和人肝细胞 WRL68，HepG2 非瘤性永生化细胞系：PH5CH，Huh7 1997年 感染性HCV cDNA 克隆分子 1999年 HCV 复制子系统

  4. structural nonstructural 5′ 3′ 2419 1711 1026 1972 1657 383 746 809 191 C p7 4A 4B 5A 5B E1 E2 2 3 metallo/Cys-proteinase RNA-dependent RNA polymerase RNA binding nucleocapsid NS3-prot. cofactor replication? ? NTPase/ helicase replication interferon resistance Envelope glycoproteins serine proteinase schematic presentation of the HCV genome

  5. HCV基因组的异质性 • 基因型(Genotype)：1型,2型,3型,4型,5型,6型 • 亚型(Subtype)：1a,1b,2a,2b,2c,3a等 50多型 • 准种(Quasispecies)：同一个体内存在着一群密切相关但不相同的变异株。

  6. HCV复制子(replicon)的发展史 HCV 1b型Con1 RNA 序列 (1999年，science) Huh-7细胞系，适应性变异 HCV 1b型HCV-N RNA 序列 其它亚型的扩展 HCV 1a型H77感染性克隆分子 HCV 1a型,2a型RNA 序列 Huh-7.5，”curing” 细胞克隆

  7. HCV复制子研究中的重要问题 • 复制子的构成 • 细胞的容受性 • 适应性变异 • 异源性序列的作用 • 完整病毒颗粒的装配

  8. Schematic representation of HCV RNAs. The first 21 amino acids of the core coding region (solid box), the gene (Neo, shaded box), the EMCV IRES (EMCV, solid line), H or Con1 indicate H77- or Con1-derived sequences. Blight kj, et al. J Virol, 2003

  9. 细胞的容受性 • Huh-7细胞 不同传代的Huh-7细胞容受性有100多倍的差异 • Huh-7.5细胞 “Curing”细胞

  10. 适应性变异的常见位点 • 集中于NS5A富含丝氨酸的中心区域 • NS3解链酶的分子表面 • NS4B的两个位点 K1846T和V1897L/M/A

  11. Location of cell culture adaptive mutations and increase of G418 transduction efficiency

  12. 30,000倍H/SG-Neo(I) 800倍H/SG-Neo(I) -7倍Con1/SG-Neo(I) -34倍Con1/SG-Neo(I) Colony-forming abilities of H77 subgenomic RNAs containing mutations in NS3. NS5A: S2204I. NS3: A1226D, P1496L Blight kj, et al. J Virol, 2003

  13. Detection of HCV proteins and RNA in Huh-7.5 cells transiently transfected with subgenomic and full-length HCV RNAs. Blight kj, et al. J Virol, 2003

  14. Effects of cell culture adaptive mutation on infectivity in vivo

  15. Replication of HCV RNAs with and without heterologous elements. Blight kj, et al. J Virol, 2003

  16. 表达NS3蛋白的细胞比例。 FSC-H forward scatter FL1-H fluorescence Blight kj, et al. J Virol, 2003

  17. HCV复制子研究现状 • H77序列复制子能在Huh-7.5细胞系中复制而不能在Huh-7亲代细胞中复制，强调细胞因素对HCV复制的重要性 • H77序列复制子至少需要两个适应性变异， S2204I，A1226D或 P1496L。Con1复制子共发现4个适应性变异：R1283G，E1383A，K1609E，K1577R。另有E1202G，T1280I。目前，这8个适应性变异中有7个位于NS3螺旋酶区域。适应性变异加强HCV体外复制的机制尚不明了 • 缺乏neo 及EMCV IRES的复制子复制效率更高。H/∆E1-P7> H/SG-5´HE >H/SG-neo 表明EMCV IRES并非HCV体外复制及复制酶表达所必需 • 不同基因型之间，亚基因组与全基因组的复制效率不一致，Con1全基因组在15%Huh-7.5细胞中复制，而亚基因组为65%；H77复制子全基因组与亚基因组复制效率是一致的，15%与18%。这种差异强调研究不同基因型复制子的重要性 • 迄今，在全基因组复制子研究中，未见HCV颗粒的装配及释放，其它基因型是否也存在这种现象有待研究

  18. HCV复制子的应用 • HCV复制相关基因结构及功能研究 • HCV与宿主细胞间相互作用的研究 • 抗HCV药物的研发及筛选

  19. HCV复制相关基因结构的研究 • 发现HCV5'-UTR在复制及翻译中的作用 • 发现HCV3'-X尾在复制的作用中 • NS5B突变对复制的影响

  20. The variable region in the 3' NTR enhances RNA replication. (A) Representative result of a transient-replication assay with luciferase eplicons carrying given engineered restriction sites in the variable region of the 3'NTR. (B) Transient replication of luciferase repliconslacking part or the complete variable region (Δvar-9401 and Δvar-9415, respectively). (C) Effects of mutations in the variable region of selectable replicons on the number of Geneticin-resistant colonies.

  21. Determination of the minimal length of the poly(U/UC) tract required for RNA replication. (A) Result of a transient-replication assay using luciferase replicons that carry a homopolymeric uridine tract of 1, 6, 26, or 46 nucleotides. (B) Number of Geneticin-resistant colonies obtained after transfection of selectable replicons with the same modifications in the 3' NTR in panel A. Representative results obtained after transfection of 100 ng of in vitro transcript are shown. (C) Sequence analysis of the poly(U/UC) tracts of replicons isolated from Huh-7 cells that had been transfected with U6 replicons and subjected to selection with Geneticin.

  22. 抗病毒药物的筛选 • 包含病毒复制所需关键酶编码基因：Proteinase、NTPase、Helicase、RdRp，潜在的抗病毒靶位 • 包含5´-UTR和3´-UTR：HCV复制翻译的关键基因 • 亚基因组复制子能在Huh7细胞系中稳定高效率复制

  23. Antiviral effects of IFN-α , Poly(I)-poly (c) ,and IFN-γa aReplicon lines were treated with IFN- α, poly(I)-poly(C), or IFN- γfor 7 to 9 days. In this experiment, clone 8 cells were treated with IFN- α, while clone 45 cells were treated with IFN- γand poly(I)-poly(C). Treatments were initiated on confluent cultures, and the medium was changed every 2 days. No overt toxicity was observed. b Replicon RNA was quantified by TaqMan RT-PCR, and values are expressed as fold reduction compared to the values for untreated cells. c The TaqMan RT-PCR assay for replicon RNA was multiplexed for the GAPDH mRNA to demonstrate a lack of effect of treatments on cellular mRNA, and all values were normalized for GAPDH. GAPDH values are expressed as Ct, the amplification cycle at which the values exceeded the background threshold.

  24. 92例患者HCV基因分型系谱图

  25. 6a 33例 1b 20.7% 1b 13例 2a 35.9% 13例 3a 3b 14例 3b 15.2% 19例 6a 2a 3a 14.1% 14.1% 92例患者HCV基因型分布图

  26. 引物设计图 3´UTR 5´UTR 5.2kb扩增 4.4kb扩增

  27. 9.5kb 9.5kb 5.2kb 5.2kb 4.4kb 4.4kb HCV 1b 型长链 RT- nested PCR及全长基因组产物电泳图

More Related