Conformational Changes of the HIV-1 Envelope Protein during Membrane Fusion Are Inhibited by the Replacement of Its Membrane-spanning Domain
Conformational Changes of the HIV-1 Envelope Protein during Membrane Fusion Are Inhibited by the Replacement of Its Membrane-spanning Domain
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DOI：
10.1074/jbc.m109.067090
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发表时间：
2010-05-07
影响因子：
4.8
通讯作者：
Matsuda, Zene
中科院分区：
文献类型：
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作者：
Kondo, Naoyuki;Miyauchi, Kosuke;Matsuda, Zene
为了帮助理解由人类免疫缺陷病毒-1（HIV-1）包膜蛋白诱导的膜融合的动态性质，我们开发了一种新的基于细胞的实时检测系统，采用一对新的报告蛋白。报告蛋白由一对与分裂绿色荧光蛋白（spGFP）融合的分裂海肾荧光素酶（spRL）组成。选择spGFP模块不仅是为了补偿spRL的弱自缔合，而且是为了在膜融合期间提供视觉报告信号。该报告基因与海肾荧光素酶的膜可渗透底物一起使用，实现了使用活细胞对膜融合的简单实时监测。我们分析了HIV-1包膜突变体，其跨膜结构域被血型糖蛋白A或水泡性口炎病毒G蛋白取代。这些突变体表现出较慢的膜融合动力学。在融合抑制剂，可溶性CD 4和C34的存在下，膜融合的分析，揭示了这些替换延长的期间，在此期间的突变体是敏感的抑制剂，与野生型相比。这些结果表明，跨膜结构域内的突变对HIV-1包膜蛋白产生变构效应，可能影响受体诱导的蛋白质胞外结构域的构象变化。
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.