A reporter cell line for rapid and sensitive evaluation of hepatitis C virus infectivity and replication
A reporter cell line for rapid and sensitive evaluation of hepatitis C virus infectivity and replication
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DOI：
10.1016/j.antiviral.2009.04.007
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发表时间：
2009-08-01
影响因子：
7.6
通讯作者：
Patel, Arvind H.
中科院分区：
文献类型：
--
作者：
Iro, Michaela;Witteveldt, Jeroen;Patel, Arvind H.
人类病原体丙型肝炎病毒（HCV）与慢性肝病有关。细胞培养感染性丙型肝炎病毒 (HCVcc) 系统的最新发展为详细研究病毒的生命周期及其与宿主细胞的相互作用开辟了途径。目前定量细胞培养物中病毒感染性的方法既耗时又费力。本研究描述了基于细胞的分泌型碱性磷酸酶 (SEAP) 报告基因测定的产生，以促进 HCV 感染和复制的体外研究。该测定基于一种新型报告细胞系，该细胞系稳定表达增强型绿色荧光蛋白 (EGFP)，通过病毒 NS3/4A 丝氨酸蛋白酶的识别序列与分泌的碱性磷酸酶框内融合。来自类似构建体的 SEAP 报告基因先前已被证明从融合蛋白中释放出来，并在被病毒 NS3/4A 蛋白酶切割后分泌到细胞外培养基中。报告细胞系能够快速、灵敏地定量细胞培养物中的 HCV 感染和病毒复制。使用HCV的几种基因型间和基因型内嵌合体证明了该系统用于研究病毒进入以及用于高通量筛选进入抑制剂和其他抗病毒化合物的效用。 (C) 2009 Elsevier B.V. 保留所有权利。
The human pathogen hepatitis C virus (HCV) is associated with chronic liver disease. The recent development of the cell culture infectious HCV (HCVcc) system has opened up avenues for detailed studies on the life cycle of the virus and its interaction with the host cell. Current methods to quantitate virus infectivity in cell culture are time-consuming and labor-intensive. This study describes the generation of a cell-based secreted alkaline phosphatase (SEAP) reporter assay to facilitate in vitro studies of HCV infection and replication. This assay is based on a novel reporter cell line stably expressing the enhanced green fluorescent protein (EGFP) fused in-frame to the secreted alkaline phosphatase via a recognition sequence of the viral NS3/4A serine protease. The SEAP reporter from a similar construct has previously been shown to be released from the fusion protein and be secreted into the extracellular culture medium following cleavage by the viral NS3/4A protease. The reporter cell line enabled rapid and sensitive quantification of HCV infection and viral replication in cell culture. The utility of this system for investigating virus entry, and for high throughput screening of entry inhibitors and other antiviral compounds was demonstrated using several inter- and intra-genotypic chimeras of HCV. (C) 2009 Elsevier B.V. All rights reserved.