Anionic Lipids Are Required for Vesicular Stomatitis Virus G Protein-mediated Single Particle Fusion with Supported Lipid Bilayers
Anionic Lipids Are Required for Vesicular Stomatitis Virus G Protein-mediated Single Particle Fusion with Supported Lipid Bilayers
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DOI：
10.1074/jbc.m113.462028
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发表时间：
2013-05-03
影响因子：
4.8
通讯作者：
Melikyan, Gregory B.
中科院分区：
文献类型：
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作者：
Matos, Pedro M.;Marin, Mariana;Melikyan, Gregory B.
病毒糖蛋白在与同源受体结合和/或经历低pH时，介导病毒和细胞膜之间的融合。尽管病毒糖蛋白的激活被认为是融合所必需的和充分的，但越来越多的证据表明，包括脂质在内的其他细胞因素可以调节融合过程。了解脂类在病毒通过内吞作用进入体内的作用受到可获得性差和内小体高度多样化的性质的阻碍。在这里，我们对单个逆转录病毒颗粒与水疱性口炎病毒(VSV)G蛋白与葡聚糖支持的脂双层的融合进行了成像。通过将可扩散的荧光标记掺入病毒膜和病毒内部，可以在低pH条件下检测VSV G介导的融合的脂质混合(半融合)和内容转移(完全融合)步骤。虽然不能检测到单病毒与两性离子脂类载体双层的融合，但阴离子脂类、磷脂酰丝氨酸和双(单酰甘油)磷酸(BMP)的加入大大提高了半融合的效率，并允许完全融合。重要的是，脂质混合总是先于融合孔的打开，这表明VSV G介导的融合是通过长寿命的半融合中间体进行的。脂质和成分转移的动力学分析表明，与含有PS的双层膜相比，含有BMP的双层膜的脂质和决定半输液中间体寿命的成分混合之间的滞后时间显着缩短。BMP是一种晚期滞留在内体的脂质，其强烈的融合促进作用与VSV在早期启动融合，但到达晚期内体隔室后将其核心释放到胞浆中的模型一致。
Viral glycoproteins mediate fusion between viral and cellular membranes upon binding to cognate receptors and/or experiencing low pH. Although activation of viral glycoproteins is thought to be necessary and sufficient for fusion, accumulating evidence suggests that additional cellular factors, including lipids, can modulate the fusion process. Understanding the role of lipids in virus entry via endocytosis is impeded by poor accessibility and the highly diverse nature of endosomes. Here we imaged fusion of single retroviral particles pseudotyped with the vesicular stomatitis virus (VSV) G protein with dextran-supported lipid bilayers. Incorporation of diffusible fluorescent labels into the viral membrane and the viral interior enabled detection of the lipid mixing (hemifusion) and content transfer (full fusion) steps of VSV G-mediated fusion at low pH. Although single virus fusion with supported bilayers made of zwitterionic lipids could not be detected, inclusion of anionic lipids, phosphatidylserine, and bis(monoacylglycero) phosphate (BMP), greatly enhanced the efficiency of hemifusion and permitted full fusion. Importantly, lipid mixing always preceded the opening of a fusion pore, demonstrating that VSV G-mediated fusion proceeds through a long-lived hemifusion intermediate. Kinetic analysis of lipid and content transfer showed that the lags between lipid and content mixing defining the lifetime of a hemifusion intermediate were significantly shorter for BMP-containing compared with PS-containing bilayers. The strong fusion-enhancing effect of BMP, a late endosome-resident lipid, is consistent with the model that VSV initiates fusion in early endosomes but releases its core into the cytosol after reaching late endosomal compartments.