Holland Anne U, Munk Carsten, Lucero Ginger R, Nguyen Lucia D, Landau Nathaniel R
Infectious Disease Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
Virology. 2004 Feb 20;319(2):343-52. doi: 10.1016/j.virol.2003.11.012.
The fusion reaction mediated by viral envelope glycoproteins proceeds through an ordered series of conformational changes in the envelope glycoprotein. Fusion inhibitors have been developed that target glycoprotein subunits, arresting the reaction at different points in the process. We report the development of a novel method for detecting viral glycoprotein-mediated fusion that is based on the principle of alpha-complementation of beta-galactosidase. The method is simple, accurate, has a high signal-to-noise ratio, is suited for high-throughput screening, and does not require new transcription or protein synthesis. Cells expressing a viral envelope glycoprotein and the N-terminal alpha fragment of beta-galactosidase were mixed with cells expressing the C-terminal beta-galactosidase fragment, CD4, CCR5, or CXCR4. Fusion was detected after 30 min and continued to increase to very high levels for more than 5 h. The assay was used to examine the temperature dependence of fusion and the effect of coreceptor and glycoprotein density on inhibitor activity.
由病毒包膜糖蛋白介导的融合反应通过包膜糖蛋白中一系列有序的构象变化进行。已经开发出针对糖蛋白亚基的融合抑制剂，可在该过程的不同点阻止反应。我们报告了一种基于β-半乳糖苷酶α-互补原理检测病毒糖蛋白介导的融合的新方法。该方法简单、准确、信噪比高，适用于高通量筛选，且不需要新的转录或蛋白质合成。将表达病毒包膜糖蛋白和β-半乳糖苷酶N端α片段的细胞与表达C端β-半乳糖苷酶片段、CD4、CCR5或CXCR4的细胞混合。30分钟后检测到融合，并在5小时以上持续增加到非常高的水平。该测定法用于检查融合的温度依赖性以及共受体和糖蛋白密度对抑制剂活性的影响。
