Bobone Sara, Hilsch Malte, Storm Julian, Dunsing Valentin, Herrmann Andreas, Chiantia Salvatore
Institute for Biology, IRI Life Sciences, Humboldt-Universität zu Berlin, Berlin, Germany.
Institute for Biochemistry and Biology, Potsdam Universität, Potsdam, Germany.
J Virol. 2017 May 26;91(12). doi: 10.1128/JVI.00267-17. Print 2017 Jun 15.
Influenza A virus matrix protein 1 (M1) is an essential component involved in the structural stability of the virus and in the budding of new virions from infected cells. A deeper understanding of the molecular basis of virion formation and the budding process is required in order to devise new therapeutic approaches. We performed a detailed investigation of the interaction between M1 and phosphatidylserine (PS) (i.e., its main binding target at the plasma membrane [PM]), as well as the distribution of PS itself, both in model membranes and in living cells. To this end, we used a combination of techniques, including Förster resonance energy transfer (FRET), confocal microscopy imaging, raster image correlation spectroscopy, and number and brightness (N&B) analysis. Our results show that PS can cluster in segregated regions in the plane of the lipid bilayer, both in model bilayers constituted of PS and phosphatidylcholine and in living cells. The viral protein M1 interacts specifically with PS-enriched domains, and such interaction in turn affects its oligomerization process. Furthermore, M1 can stabilize PS domains, as observed in model membranes. For living cells, the presence of PS clusters is suggested by N&B experiments monitoring the clustering of the PS sensor lactadherin. Also, colocalization between M1 and a fluorescent PS probe suggest that, in infected cells, the matrix protein can specifically bind to the regions of PM in which PS is clustered. Taken together, our observations provide novel evidence regarding the role of PS-rich domains in tuning M1-lipid and M1-M1 interactions at the PM of infected cells. Influenza virus particles assemble at the plasma membranes (PM) of infected cells. This process is orchestrated by the matrix protein M1, which interacts with membrane lipids while binding to the other proteins and genetic material of the virus. Despite its importance, the initial step in virus assembly (i.e., M1-lipid interaction) is still not well understood. In this work, we show that phosphatidylserine can form lipid domains in physical models of the inner leaflet of the PM. Furthermore, the spatial organization of PS in the plane of the bilayer modulates M1-M1 interactions. Finally, we show that PS domains appear to be present in the PM of living cells and that M1 seems to display a high affinity for them.
甲型流感病毒基质蛋白1（M1）是参与病毒结构稳定性以及新病毒粒子从感染细胞中出芽过程的重要组成部分。为了设计新的治疗方法，需要更深入地了解病毒粒子形成和出芽过程的分子基础。我们对M1与磷脂酰丝氨酸（PS）（即其在质膜[PM]上的主要结合靶点）之间的相互作用以及PS本身在模型膜和活细胞中的分布进行了详细研究。为此，我们结合使用了多种技术，包括荧光共振能量转移（FRET）、共聚焦显微镜成像、光栅图像相关光谱以及数量与亮度（N&B）分析。我们的结果表明，在由PS和磷脂酰胆碱构成的模型双层膜以及活细胞中，PS能够在脂质双层平面内的隔离区域聚集。病毒蛋白M1与富含PS的结构域特异性相互作用，这种相互作用进而影响其寡聚化过程。此外，正如在模型膜中观察到的那样，M1能够稳定PS结构域。对于活细胞，通过监测PS传感器乳凝集素聚集的N&B实验表明存在PS簇。而且，M1与荧光PS探针的共定位表明，在感染细胞中，基质蛋白能够特异性结合到PS聚集的质膜区域。综上所述，我们的观察结果为富含PS的结构域在调节感染细胞质膜上的M1-脂质和M1-M1相互作用中的作用提供了新的证据。流感病毒粒子在感染细胞的质膜（PM）上组装。这个过程由基质蛋白M1精心编排，M1在与病毒的其他蛋白质和遗传物质结合的同时与膜脂相互作用。尽管其很重要，但病毒组装的初始步骤（即M1-脂质相互作用）仍未得到很好的理解。在这项工作中，我们表明磷脂酰丝氨酸能够在质膜内小叶的物理模型中形成脂质结构域。此外，双层膜平面内PS的空间组织调节M1-M1相互作用。最后，我们表明PS结构域似乎存在于活细胞的质膜中，并且M1似乎对它们具有高亲和力。
