Quantitative analysis of the molecular interactions driving Influenza A virus assembly in model and cellular membranes

细胞膜是复杂的生物实体，具有广泛的功能。例如，质膜(PM)中脂质和受体的侧向组织参与了几个生物过程，如细胞间通讯或病毒感染。在这种情况下，流感病毒(IV)感染一个细胞并最终传播到其他细胞的机制是典型的。事实上，已知的是，子代流感病毒粒子的组装和萌发是一个复杂的过程，通过PM的特殊脂蛋白结构域发生，称为RAFT。不幸的是，这一过程背后的详细分子机制尚不清楚。为了剖析病毒组分与宿主细胞的脂质和膜蛋白之间的复杂相互作用，我们将使用新的PM物理模型，即不对称模型膜。这代表着膜生物物理学领域向前迈出的重要一步，因为直到最近，脂蛋白相互作用的研究几乎完全是在对称的简单细胞膜模型上进行的。我们的总体目标是通过重组上述可靠和可控的膜模型中的关键病毒成分，并结合原子力显微镜和先进的荧光显微镜技术来表征它们。首先，我们将重点关注病毒基质蛋白M1在协调IV组分的空间组装形成新的病毒芽中的作用此外，我们还将研究PM和IV感染周期中RAFT结构域之间的关系。事实上，IV组分和PM RAFT结构域之间的相互作用还没有被很好地理解。一方面，我们将定量地表征IV蛋白与高级(即不对称)的RAFT结构域模型之间的相互作用。另一方面，对于PM的新的不对称模型的使用将使我们有机会从总体上了解PM结构域的结构是如何受到小叶成分不对称和小叶之间的耦合的影响的。这一主题在膜生物物理学领域具有特别重要的意义，因为有序的PM结构域参与了几个生理过程，如细胞信号传递。从这项建议中描述的实验中获得的知识将对了解IV从宿主细胞中退出的分子机制以及最终开发防止病毒感染传播的创新方法具有重要价值。

Cellular membranes are complex biological entities which perform a wide range of functions. The lateral organization of lipids and receptors in the plasma membrane (PM), for example, is involved in several biologicalprocesses such as cell-cell communication or viral infections. Themechanism by which the Influenza virus (IV) infects a cell and, eventually,spreads to other cells is paradigmatic in this context. It is known, in fact, that assembly and budding of progeny influenza virions is an intricate process that occurs through specialized lipid-protein domains of the PM named rafts.Unfortunately, the detailed molecular mechanisms behind this processremain unclear. In order to dissect the complex interactions between viralcomponents and the lipids and membrane proteins of the host cell, we will use novel physical models of the PM, i.e. asymmetric model membranes. This represents an important step forward in the field of membrane biophysics since, until recently, investigation of lipid-protein interaction has been carried almost exclusively on symmetric simple models of cellular membranes. These model bilayers did not take into account the important compositional asymmetry between inner and outer leaflets of PM.Our general goal is to clarify the molecular interactions governing thebudding and exit of IV from infected cells, by reconstituting the key viralcomponents in the above-mentioned reliable and controlled membranemodels and characterizing them with a sophisticated combination of Atomic Force Microscopy and advanced fluorescence microscopy techniques.Firstly, we will focus on the role of the viral matrix protein M1 in orchestrating the spatial assembly of IV components into a new virion budding from the host PM. Furthermore, we will investigate the relationship between the raft domains in the PM and IV infectious cycle. The interaction between IV components and PM raft domains is, in fact, not well understood. On one hand, we will quantitatively characterize the interplay between IV proteins and advanced (i.e. asymmetric) models of raft domains. On the other hand, the use of novel asymmetric models for the PM will provide us the chance to understand in general how the structure of PM domains is affected by leaflet compositional asymmetry and by the coupling between leaflets. This topic is of particular importance in the field of membrane biophysics, since orderedPM domains are involved in several physiological processes, such as cell signaling. The knowledge deriving from the experiments described in this proposal will be of great value for understanding the molecular mechanisms of IV exit from host cells and, ultimately, developing innovative methods to prevent the spreading of the viral infection.