Lateral sorting of proteins into lipid-rafts and protein-protein interactions as prerequisite for assembly of influenza virus: A complementary biophysical approach on model membranes and living cells

被包裹的病毒的组装和萌发需要在宿主膜的萌发部位局部浓缩病毒成分。有两种非排斥机制可以解释这一现象：膜蛋白与质膜亚区(脂筏)的结合以及其他病毒成分与膜蛋白的结合，以及病毒蛋白之间的特定相互作用。病毒萌发的脂筏概念基于两个观察结果：(1)许多病毒的膜蛋白存在于脂筏中；(2)脂筏的解体抑制了病毒颗粒的组装。然而，已发表的证明病毒蛋白存在于木筏中的结果是通过Triton提取方法获得的，并且蛋白质被分配到这些耐洗涤剂的膜上是否反映了它们与活细胞内的木筏的联系仍存在很大争议。此外，分子基础，即将病毒蛋白质分选到脂类结构域的信号是一个悬而未决的问题。同样，糖蛋白和病毒颗粒内部成分之间的相互作用只从遗传实验中间接推断出来，但没有在细胞内直接证明。在这个项目中，我们将使用基本的生物物理方法解决蛋白质在RAFT中的分配以及病毒蛋白质之间的相互作用：？已建立的RAFT标记与病毒蛋白质之间以及细胞内两种病毒蛋白质之间的荧光能量转移(FRET)。？将病毒膜蛋白重组为含有液体无序和液体有序的类木筏相的巨型单胺Har小泡(GUV)。跨膜结构域序列与脂类之间的特异性相互作用。我们将分析流感病毒膜蛋白血凝素(HA)、神经氨酸酶(NA)和基质蛋白(ML)。根据Triton提取实验，NA和HA是脂筏的成分，而ML只有在NA或HA存在的情况下才与脂筏结合。我们还将讨论是否可以使用人工系统作为GUV来模拟与病毒萌发相关的膜弯曲过程，并确定触发膜弯曲所必需的病毒成分。然而，所提出的方法适用于分析任何病毒蛋白的RAFT-亲和力和蛋白质-蛋白质-相互作用，并将在Schwerpenkt努力将我们的方法应用于其他包膜病毒。

Assembly and budding of enveloped viruses requires local enrichment of viral components at the budding site of the host membranes. Two non-excluding mechanisms can account for this: inclusion of membrane proteins into and association of other viral components with subdomains of the plasma membrane (lipid-rafts), and specific interactions between viral proteins. The lipid-raft concept for virus budding is based on two observations: (i) membrane proteins of many viruses are present in rafts and (ii) disintegration of rafts inhibits assembly of virus particles. However, published results demonstrating the occurrence of viral proteins in rafts were obtained with the Triton-extraction method and it is highly controversial whether partitioning of proteins into these detergent-resistant membranes reflects their association with rafts inside living cells. Furthermore, the molecular basis, i.e. the signal for sorting a viral protein into a lipid domain is an open question. Likewise, interactions between glycoproteins and internal components of virus particles were only indirectly deduced from genetic experiments, but have not been directly demonstrated inside cells. In this project we will address partitioning of proteins into rafts and interactions between viral proteins using essentially biophysical approaches: ¿ Fluorescence-energy transfer (FRET) between established raft-markers and viral proteins as well as between two viral proteins inside cells. ¿ Reconstitution of viral membrane proteins into giant unilameHar vesicles (GUVs) harbouring a liquid-disordered and a liquid-ordered, raft-like phase. ¿ Specific interaction between transmembrane domain sequences and lipids. We will analyze the well-characterized influenza virus membrane proteins hemagglutinin (HA), neuraminidase (NA) and the matrix-protein (Ml). Based on Triton-extraction experiments NA and HA are constituents of lipid-rafts, whereas Ml binds to rafts only when either NA or HA are present. We will also address whether artificial systems as GUVs can be used to model membrane bending processes associated with virus budding, and to identify the viral components necessary to trigger membrane bending. However, the proposed methods are suitable to analyze the raft-affinities and protein-protein-interactions for any viral protein, and efforts will be undertaken in the Schwerpunkt to apply our approach to other enveloped viruses.