Inhibition of hepatitis C virus p7 membrane channels in a liposome-based assay system
Inhibition of hepatitis C virus p7 membrane channels in a liposome-based assay system
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DOI：
10.1016/j.antiviral.2007.05.001
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发表时间：
2007-10-01
影响因子：
7.6
通讯作者：
Griffin, Stephen
中科院分区：
文献类型：
--
作者：
StGelais, Corine;Tuthill, Tobias J.;Griffin, Stephen
慢性丙型肝炎病毒（HCV）患者的化疗在50%以上的病例中无效，对新药物靶点产生了很高的需求。HCV的p7蛋白在体外表现出膜通道活性，对体内复制至关重要，尽管其在病毒生命周期中的确切作用尚不清楚。P7通道活性可以被几种化合物特异性抑制，使该蛋白成为药物开发的有吸引力的候选蛋白，尽管迄今为止用于表征该蛋白的技术不适合化合物文库筛选。在这里，我们描述了一种基于从脂质体释放荧光指示剂的p7通道形成能力的测定。我们发现来自基因型1b HCV的重组p7在与脂质体混合时引起染料的剂量依赖性释放，并且这种特性在酸性ph下增强。我们证明这种活性是由于形成大小选择性孔而不是脂质体的非特异性破坏，并且这种活性可以被金刚烷胺和其他几种化合物阻断，验证其作为p7通道功能的测量。该系统为开发p7作为治疗靶点提供了第一个方便的体外试验。(c) 2007 Elsevier B.V.版权所有
Chemotherapy for patients chronically infected with hepatitis C virus (HCV) is ineffective in over 50% of cases, generating a high demand for new drug targets. The p7 protein of HCV displays membrane channel activity in vitro and is essential for replication in vivo though its precise role in the virus life cycle is unknown. p7 channel activity can be specifically inhibited by several classes of compounds, making this protein an attractive candidate for drug development, though techniques used to date in characterising this protein are unsuited to compound library screening. Here we describe an assay for the channel forming ability of p7 based on the release of a fluorescent indicator from liposomes. We show that recombinant p7 from genotype 1b HCV causes a dose-dependent release of dye when mixed with liposomes and that this property is enhanced at acidic pH. We demonstrate that this activity is due to the formation of a size-selective pore rather than non-specific disruption of liposomes and that activity can be blocked by amantadine and several other compounds, validating it as a measure of p7 channel function. This system provides the first convenient in vitro assay for exploiting p7 as a therapeutic target. (c) 2007 Elsevier B.V. All rights reserved.