Tibotec BVBA, 2800 Mechelen, Belgium.
J Virol Methods. 2010 May;165(2):268-76. doi: 10.1016/j.jviromet.2010.02.007. Epub 2010 Feb 11.
The hepatitis C virus (HCV) replicates its genome on a membrane-associated replication complex. These complexes are represented by "dot-like" structures on the endoplasmic reticulum when standard fluorescence microscopy techniques are applied. To screen compound libraries for inhibitors interfering with the formation of the HCV replication complex independent of RNA replication, an image-based high-content screening assay was developed utilizing inducible expression of the HCV non-structural proteins NS3-5B in an U2-OS Tet-On cell line. An eGFP was fused to NS5A for the detection of replication complexes. The cell line was tightly regulated and the eGFP insertion within NS5A did not alter polyprotein processing. The NS5AeGFP signal colocalized with other non-structural proteins in "dot-like" structures. Accompanying image analysis tools were developed enabling the detection of changes in replication complex formation. Finally, the addition of a HCV NS3/4A protease inhibitor resulted in a dose-dependent reduction of "dot-like" structures demonstrating the practicability of the assay.
丙型肝炎病毒 (HCV) 在膜相关复制复合物上复制其基因组。当应用标准荧光显微镜技术时，这些复合物表现为内质网上的“点状”结构。为了筛选抑制 HCV 复制复合物形成而不影响 RNA 复制的化合物库，利用 HCV 非结构蛋白 NS3-5B 在 U2-OS Tet-On 细胞系中的诱导表达，开发了一种基于图像的高通量筛选测定法。将 eGFP 融合到 NS5A 中用于检测复制复合物。该细胞系受到严格调控，并且 NS5A 内的 eGFP 插入不改变多蛋白加工。NS5AeGFP 信号与“点状”结构中的其他非结构蛋白共定位。开发了伴随的图像分析工具，能够检测复制复合物形成的变化。最后，添加 HCV NS3/4A 蛋白酶抑制剂会导致“点状”结构的数量减少，证明了该测定法的实用性。
