Developing Efficient HCV Replication Cell Culture System

开发高效的 HCV 复制细胞培养系统

• 批准号: 6557819
• 负责人: Shinji Makino
• 金额: $ 7.45万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-30 至 2004-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6557819
• 关键词: cell line; hepatitis C virus; technology /technique development; tissue /cell culture; virus RNA; virus replication

DESCRIPTION (provided by applicant): No cell culture system exists that efficiently supports hepatitis C virus (HCV) replication and assembly; cloned, full-length HCV RNA cannot replicate efficiently and assemble into viral particles in today's cell lines. One cell line, Huh-7 cell line, however, supports continuous replication of HCV replicons. In an HCV replicon, NNeo, the neomycin phosphotransferase gene and the encephalomyocarditis virus internal ribosome entry site sequence replace the HCV structural protein genes and NS2 gene. NNeo RNA efficiently accumulated within the first 3 days after transfection of in vitro-synthesized NNeo RNA transcripts into Huh-7 cells. In marked contrast, full-length HCV RNA did not efficiently accumulate after transfection of the cloned, infectious RNA transcripts that are the replicon's parent. Determining the reason the full-length HCV RNA did not replicate well in the cell culture is at the heart of establishing an efficient HCV replication system in cell culture. The present application aims to understand why only a low level of RNA synthesis occurs after transfection of full-length HCV in Huh-7 cells. We will test whether replication of full-length HCV may induce cell death, thereby suppressing HCV RNA replication. We will examine whether the enormity of full-length HCV RNA might severely affect its replication efficiency. Furthermore, the possibility that an RNA element(s) that suppresses HCV RNA replication may be encoded in full-length HCV RNA will be explored. Information from the studies proposed here will answer why the full-length HCV clone does not replicate well in Huh 7 cells, and will let us reach our ultimate aim of developing an efficient HCV replication system in cell culture.

描述（由申请人提供）：不存在有效支持丙型肝炎病毒（HCV）复制和组装的细胞培养系统；克隆的全长 HCV RNA 无法在当今的细胞系中有效复制并组装成病毒颗粒。然而，一种细胞系 Huh-7 细胞系支持 HCV 复制子的连续复制。在HCV复制子中，NNeo、新霉素磷酸转移酶基因和脑心肌炎病毒内部核糖体进入位点序列取代了HCV结构蛋白基因和NS2基因。将体外合成的 NNeo RNA 转录物转染至 Huh-7 细胞后的前 3 天内，NNeo RNA 有效积累。与此形成鲜明对比的是，在转染作为复制子亲本的克隆感染性RNA转录物后，全长HCV RNA并未有效积累。确定全长 HCV RNA 在细胞培养物中复制不佳的原因是在细胞培养物中建立高效 HCV 复制系统的核心。本申请旨在了解为什么在Huh-7细胞中转染全长HCV后仅发生低水平的RNA合成。我们将测试全长HCV的复制是否会诱导细胞死亡，从而抑制HCV RNA复制。我们将研究全长 HCV RNA 的巨大是否会严重影响其复制效率。此外，将探索抑制HCV RNA复制的RNA元件可能被编码在全长HCV RNA中的可能性。这里提出的研究信息将回答为什么全长 HCV 克隆在 Huh 7 细胞中不能很好地复制，并使我们达到在细胞培养中开发高效 HCV 复制系统的最终目标。