Membrane fusion induced by vesicular stomatitis virus depends on histidine protonation
Membrane fusion induced by vesicular stomatitis virus depends on histidine protonation
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DOI：
10.1074/jbc.m210615200
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发表时间：
2003-04-18
影响因子：
4.8
通讯作者：
Da Poian, AT
中科院分区：
文献类型：
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作者：
Carneiro, FA;Stauffer, F;Da Poian, AT
包膜动物病毒进入宿主细胞总是依赖于由病毒包膜糖蛋白构象变化引发的膜融合步骤。水疱性口炎病毒（VSV）感染是由病毒刺突糖蛋白G介导的，它在酸性环境中诱导膜融合。vsv诱导的膜融合发生在非常窄的pH范围内，在6.2和5.8之间，表明His质子化。是此过程所必需的。为了研究His在VSV融合中的作用，我们用焦碳酸二乙基（DEPC）对这些残基进行了化学修饰。我们发现DEPC处理以浓度依赖的方式抑制VSV介导的膜融合，并且将修饰的病毒与羟胺孵育完全逆转融合的完全抑制。荧光测量显示，用DEPC修饰VSV消除了ph诱导的G蛋白构象变化，表明在酸性ph下，His质子化驱动G蛋白与靶膜相互作用。质谱分析修饰G蛋白的色氨酸片段可以鉴定推定的活性His残基。通过合成多肽，我们发现DEPC修饰His-148和His-149，并用Ala取代这些残基，完全抑制了肽诱导的融合，这表明这些His直接参与了VSV融合。
Entry of enveloped animal viruses into their host cells always depends on a step of membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. VSV-induced membrane fusion occurs at a very narrow pH range, between 6.2 and 5.8, suggesting that His protonation. is required for this process. To investigate the role of His in VSV fusion, we chemically modified these residues using diethylpyrocarbonate (DEPC). We found that DEPC treatment inhibited membrane fusion mediated by VSV in a concentration-dependent manner and that the complete inhibition of fusion was fully reversed by incubation of modified virus with hydroxylamine. Fluorescence measurements showed that VSV modification with DEPC abolished pH-induced conformational changes in G protein, suggesting that His protonation drives G protein interaction with the target membrane at acidic pH. Mass spectrometry analysis of tryptic fragments of modified G protein allowed the identification of the putative active His residues. Using synthetic peptides, we showed that the modification of His-148 and His-149 by DEPC, as well as the substitution of these residues by Ala, completely inhibited peptide-induced fusion, suggesting the direct participation of these His in VSV fusion.