SEC14L2 enables pan-genotype HCV replication in cell culture.
SEC14L2 enables pan-genotype HCV replication in cell culture.
复制标题
DOI：
10.1038/nature14899
复制
发表时间：
2015-08-27
期刊：
影响因子：
64.8
通讯作者：
Rice CM
中科院分区：
文献类型：
--
作者：
Saeed M;Andreo U;Chung HY;Espiritu C;Branch AD;Silva JM;Rice CM
自1989年发现以来，在细胞培养中培养丙型肝炎病毒（HCV）临床分离株的努力取得了有限的成功。只有JFH-1分离株具有在培养的肝癌细胞中有效复制的能力，而没有细胞培养适应性突变。我们假设培养的细胞缺乏一个或多个复制临床分离株所需的因子。为了鉴定缺失的因子，我们用合并的基于慢病毒的人cDNA文库转导Huh-7.5人肝癌细胞，用缺乏适应性突变的HCV亚基因组复制子转染，并选择稳定的复制子集落。这导致了一个单一的cDNA，SEC 14 L2，其表达允许在几个肝癌细胞系中的所有HCV基因型的RNA复制的鉴定。这种作用是剂量依赖性的，并且需要SEC 14 L2的持续存在。全长HCV基因组也复制并产生低水平的感染性病毒。值得注意的是，SEC 14 L2表达Huh-7.5细胞也支持HCV复制后接种患者血清。机制研究表明，SEC 14 L2通过增强维生素E介导的抗脂质过氧化保护作用来促进HCV感染。这为所有HCV分离株的体外复制系统的开发奠定了基础，并提供了一个有吸引力的平台来剖析细胞培养适应性突变的作用机制。
Since its discovery in 1989, efforts to grow clinical isolates of the hepatitis C virus (HCV) in cell culture have met with limited success. Only the JFH-1 isolate has the capacity to replicate efficiently in cultured hepatoma cells without cell culture-adaptive mutations. We hypothesized that cultured cells lack one or more factors required for the replication of clinical isolates. To identify the missing factors, we transduced Huh-7.5 human hepatoma cells with a pooled lentivirus-based human cDNA library, transfected with HCV subgenomic replicons lacking adaptive mutations, and selected for stable replicon colonies. This led to the identification of a single cDNA, SEC14L2, whose expression allowed RNA replication of all HCV genotypes in several hepatoma cell lines. This effect was dose-dependent, and required the continuous presence of SEC14L2. Full-length HCV genomes also replicated and produced low levels of infectious virus. Remarkably, SEC14L2-expressing Huh-7.5 cells also supported HCV replication following inoculation with patient sera. Mechanistic studies suggest that SEC14L2 promotes HCV infection by enhancing vitamin E-mediated protection against lipid peroxidation. This sets the stage for development of in vitro replication systems for all HCV isolates, and provides an attractive platform to dissect the mechanisms by which cell culture-adaptive mutations act.