Gervais Christian, Dô Florence, Cantin Ariane, Kukolj George, White Peter W, Gauthier Annick, Vaillancourt Frédéric H
Department of Biological Sciences, Research and Development, Boehringer Ingelheim (Canada) Ltd., Laval, QC, Canada.
J Biomol Screen. 2011 Mar;16(3):363-9. doi: 10.1177/1087057110396215. Epub 2011 Feb 22.
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.
丙型肝炎病毒（HCV）的p7蛋白不参与病毒RNA复制，但对传染性病毒的产生至关重要。基于其假定的离子通道活性，p7属于一类被称为病毒离子通道蛋白的病毒蛋白家族，该家族蛋白在插入脂质膜后会发生寡聚化。为了筛选能够干扰p7通道功能的化合物，对一种基于脂质体的低通量荧光染料渗透性测定方法进行了改进，并将其转化为一种强大的高通量筛选测定方法。表达重组p7的大肠杆菌在高密度补料分批发酵中生长，随后使用亲和色谱和反相色谱相结合的方法进行无去污剂纯化。脂质体的磷脂组成针对p7识别和长期稳定性进行了优化。利用蜂毒溶血肽开发了一种反筛选方法，以消除非特异性筛选命中物。基于p7脂质体的测定方法显示出强大的统计学特征（Z' > 0.75），并且使用已知抑制剂证实了其对抑制的敏感性。
