Polyak S J, Paschal D M, McArdle S, Gale M J, Moradpour D, Gretch D R
Department of Laboratory Medicine, University of Washington, Seattle, WA, USA.
Hepatology. 1999 Apr;29(4):1262-71. doi: 10.1002/hep.510290438.
The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy in clinical studies. Biochemical studies have demonstrated that NS5A interacts in vitro with and inhibits the IFN-induced, RNA-dependent protein kinase, PKR, and that NS5A interacts with at least one other cellular kinase. The present study describes the establishment and characterization of various stable NS5A-expressing human cell lines, and the development of a cell culture-based assay for determining the inherent IFN resistance of clinical NS5A isolates. Human epithelioid (Hela) and osteosarcoma (U2-OS) cell lines were generated that express NS5A under tight regulation by the tetracycline-dependent promoter. Maximal expression of NS5A occurred at 48 hours following the removal of tetracycline from the culture medium. The half-life of NS5A in these cell lines was between 4 to 6 hours. NS5A protein expression was localized cytoplasmically, with a staining pattern consistent with the location of the Golgi apparatus and endoplasmic reticulum. In the majority of cell lines, no obvious phenotypic changes were observed. However, three genotype 1b NS5A-expressing osteosarcoma cell lines exhibited cytopathic effect and severely reduced proliferation as a result of high-level NS5A expression. Full-length NS5A protein isolated from a genotype 1b IFN-nonresponsive patient (NS5A-1b) was capable of rescuing encephalomyocardititis virus replication during IFN challenge up to 40-fold, whereas a full-length NS5A-1a and an interferon sensitivity determining region (ISDR) deletion mutant (NS5A-1a-triangle upISDR) isolated from a genotype 1a IFN-nonresponsive patient showed no rescue activity. The NS5A-1b and NS5A-1a proteins also rescued vesicular stomatitis virus replication during IFN treatment by two- to threefold. These data cummulatively suggest that NS5A expression alone can render cells partially resistant to the effects of IFN against IFN-sensitive viruses, and that in some systems, these effects may be independent of the putative ISDR. A scenario is discussed in which the NS5A protein may employ multiple strategies contributing to IFN resistance during HCV infection.
丙型肝炎病毒（HCV）非结构5A（NS5A）蛋白在临床研究中与HCV对干扰素（IFN）抗病毒治疗的固有抗性有关。生化研究表明，NS5A在体外与IFN诱导的RNA依赖性蛋白激酶PKR相互作用并抑制其活性，并且NS5A与至少一种其他细胞激酶相互作用。本研究描述了各种稳定表达NS5A的人细胞系的建立和表征，以及一种基于细胞培养的测定方法的开发，用于确定临床NS5A分离株的固有IFN抗性。构建了人上皮样（Hela）和骨肉瘤（U2-OS）细胞系，它们在四环素依赖性启动子的严格调控下表达NS5A。从培养基中去除四环素后48小时，NS5A达到最大表达。这些细胞系中NS5A的半衰期在4至6小时之间。NS5A蛋白表达定位于细胞质中，染色模式与高尔基体和内质网的位置一致。在大多数细胞系中，未观察到明显的表型变化。然而，三个表达1b基因型NS5A的骨肉瘤细胞系由于高水平的NS5A表达而表现出细胞病变效应并严重降低增殖。从1b基因型IFN无反应患者分离的全长NS5A蛋白（NS5A-1b）在IFN攻击期间能够拯救脑心肌炎病毒复制达40倍，而从1a基因型IFN无反应患者分离的全长NS5A-1a和干扰素敏感性决定区（ISDR）缺失突变体（NS5A-1a-ΔISDR）没有拯救活性。NS5A-1b和NS5A-1a蛋白在IFN治疗期间也使水疱性口炎病毒复制增加了两到三倍。这些数据累积表明，单独的NS5A表达可使细胞对IFN对IFN敏感病毒的作用产生部分抗性，并且在某些系统中，这些作用可能独立于假定的ISDR。本文讨论了一种情况，即NS5A蛋白在HCV感染期间可能采用多种策略导致IFN抗性。
