Development and Validation of a High-Throughput Screening Assay for the Hepatitis C Virus p7 Viroporin
Development and Validation of a High-Throughput Screening Assay for the Hepatitis C Virus p7 Viroporin
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DOI：
10.1177/1087057110396215
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发表时间：
2011-03-01
影响因子：
--
通讯作者：
Vailancourt, Frederic H.
中科院分区：
文献类型：
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作者：
Gervais, Christian;Do, Florence;Vailancourt, Frederic H.
HCV p7蛋白不参与病毒RNA复制，但对于感染性病毒的产生是必需的。基于其推定的离子通道活性，p7属于称为病毒孔蛋白的病毒蛋白家族，其在插入脂质膜后寡聚化。为了筛选能够干扰p7通道功能的化合物，对基于低通量脂质体的荧光染料渗透性测定进行了修改，并将其转化为稳健的高通量筛选测定。表达重组p7的大肠杆菌在高密度补料分批发酵中生长，然后使用亲和和反相色谱的组合进行无洗涤剂纯化。脂质体的磷脂组成针对p7识别和长期稳定性进行了优化。使用蜂毒肽通道形成肽开发反筛选以消除非特异性筛选命中。基于p7脂质体的测定显示出稳健的统计学（Z' > 0.75），并且使用已知的抑制剂证实了对抑制的敏感性。（Journal of Biomolecular Screening 2011;16：363-369）
The HCV p7 protein is not involved in viral RNA replication but is essential for production of infectious virus. Based on its putative ion channel activity, p7 belongs to a family of viral proteins known as viroporins that oligomerize after insertion into a lipid membrane. To screen for compounds capable of interfering with p7 channel function, a low-throughput liposome-based fluorescent dye permeability assay was modified and converted to a robust high-throughput screening assay. Escherichia coli expressing recombinant p7 were grown in high-density fed-batch fermentation followed by a detergent-free purification using a combination of affinity and reversed-phase chromatography. The phospholipid composition of the liposomes was optimized for both p7 recognition and long-term stability. A counterscreen was developed using the melittin channel-forming peptide to eliminate nonspecific screening hits. The p7 liposome-based assay displayed robust statistics (Z' > 0.75), and sensitivity to inhibition was confirmed using known inhibitors. (Journal of Biomolecular Screening 2011;16:363-369)