Ochsenbauer-Jambor Christina, Jones Jennifer, Heil Marintha, Zammit Kenneth P, Kutsch Olaf
University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Biotechniques. 2006 Jan;40(1):91-100. doi: 10.2144/000112072.
The rapid increase of viral strains that are resistant to the currently available antiretroviral drugs is a threat to the success of current human immunodeficiency virus type 1 (HIV-1) treatment and emphasizes the importance of developing novel anti-HIV-1 compounds. To improve the current abilities to screen for novel HIV-1 inhibitors, here we introduce a T-cell-based reporter cell line (JLTRG-RS) that expresses both HIV-1 coreceptors, CXCR4 and CCRS, and provides the convenience of using enhanced green fluorescent protein (EGFP) as a direct and quantitative marker. Unlike previous EGFP-based reporter cell lines, JLTRG-RS cells have an unusually high dynamic signal range, sufficient for plate reader detection using a 384-well format. In this format, JLTRG-R5 cell-based infectivity assays have a Z'-factor of 0.78, which defines the assay as extremely robust and clearly amenable to high-throughput screening. The functional similarity of the JLTRG-R5 cell line and peripheral blood mononuclear cells (PBMCs) was demonstrated through the identity of the inhibitory concentrations, 50% (IC50s) for four antiretroviral compounds or neutralizing antibodies. Because EGFP can be directly and continuously quantified in cell culture, the reporter cell line requires no manipulation during assay preparation or analysis. In addition, the EGFP marker allows for data acquisition at an optimal time point by prescreening selected positive control wells using fluorescent microscopy. These characteristics make the system extremely flexible, rapid, and inexpensive. Due to its intrinsic flexibility, the JLTRG-R5 cell-based reporter system provides a powerful tool to greatly facilitate future screening for HIV-1 inhibitors.
对目前可用抗逆转录病毒药物产生耐药性的病毒株迅速增加，这对当前1型人类免疫缺陷病毒（HIV-1）治疗的成功构成威胁，并凸显了开发新型抗HIV-1化合物的重要性。为了提高目前筛选新型HIV-1抑制剂的能力，我们在此引入一种基于T细胞的报告细胞系（JLTRG-RS），该细胞系同时表达HIV-1共受体CXCR4和CCR5，并提供了使用增强型绿色荧光蛋白（EGFP）作为直接定量标记的便利。与以往基于EGFP的报告细胞系不同，JLTRG-RS细胞具有异常高的动态信号范围，足以使用384孔板格式进行酶标仪检测。在这种格式下，基于JLTRG-R5细胞的感染性测定的Z'因子为0.78，这表明该测定极其稳健，显然适用于高通量筛选。通过四种抗逆转录病毒化合物或中和抗体的50%抑制浓度（IC50）的一致性，证明了JLTRG-R5细胞系与外周血单个核细胞（PBMC）的功能相似性。由于EGFP可以在细胞培养中直接连续定量，该报告细胞系在测定准备或分析过程中无需操作。此外，EGFP标记允许通过使用荧光显微镜对选定的阳性对照孔进行预筛选，在最佳时间点采集数据。这些特性使该系统极其灵活、快速且成本低廉。由于其固有的灵活性，基于JLTRG-R5细胞的报告系统提供了一个强大的工具，极大地便于未来筛选HIV-1抑制剂。
