Research Center for Asian Infectious Diseases, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
J Biol Chem. 2010 May 7;285(19):14681-8. doi: 10.1074/jbc.M109.067090. Epub 2010 Mar 2.
To help understand the dynamic nature of membrane fusion induced by the human immunodeficiency virus-1 (HIV-1) envelope protein, we developed a new cell-based real-time assay system employing a pair of novel reporter proteins. The reporter proteins consist of a pair of split Renilla luciferase (spRL) fused to split green fluorescent protein (spGFP). The spGFP modules were chosen not only to compensate weak self-association of spRL but also to provide visual reporter signals during membrane fusion. Use of this reporter together with a membrane permeable substrate for Renilla luciferase achieved a simple real-time monitoring of membrane fusion using live cells. We analyzed the HIV-1 envelope mutants whose membrane-spanning domains were replaced with that of glycophorin A or vesicular stomatitis virus G-protein. These mutants showed a slower kinetics of membrane fusion. The analysis of membrane fusion in the presence of fusion inhibitors, soluble CD4 and C34, revealed that these replacements prolonged the period during which the mutants were sensitive to the inhibitors, as compared with the wild type. These results suggest that the mutations within the membrane-spanning domains exerted an allosteric effect on the HIV-1 envelope protein, probably affecting the receptor-induced conformational changes of the ectodomain of the protein.
为了帮助理解人类免疫缺陷病毒 1 型（HIV-1）包膜蛋白诱导的膜融合的动态特性，我们开发了一种新的基于细胞的实时分析系统，该系统采用了一对新型报告蛋白。报告蛋白由一对融合到分裂绿色荧光蛋白（spGFP）的分裂萤光素酶（spRL）组成。选择 spGFP 模块不仅可以补偿 spRL 的弱自缔合，还可以在膜融合过程中提供可视报告信号。使用这种报告蛋白和膜透性萤光素酶的底物，可以对活细胞中的膜融合进行简单的实时监测。我们分析了将其跨膜结构域替换为血影蛋白 A 或水疱性口炎病毒 G 蛋白的 HIV-1 包膜突变体。这些突变体显示出较慢的膜融合动力学。在融合抑制剂、可溶性 CD4 和 C34 的存在下分析膜融合表明，与野生型相比，这些取代物延长了突变体对抑制剂敏感的时间。这些结果表明，跨膜结构域内的突变对 HIV-1 包膜蛋白产生了别构效应，可能影响了蛋白的外域受受体诱导的构象变化。
