Paramyxovirus F protein mediated membrane fusion

描述(申请人提供)：过去十年中令人信服的工作表明，跨膜结构域(TMD)，曾经被认为仅仅作为疏水锚起作用，是驱动细胞蛋白质齐聚、细胞信号和通道调节的中心角色 功能。然而，TMDs在病毒糖蛋白稳定性和功能中的作用仍然知之甚少。在之前的资助期间，我们对Hendra病毒(HEV)融合(F)蛋白的研究确定了TMDs在调节F蛋白稳定性、细胞转运和ad融合功能中的关键作用。我们的新研究利用沉降平衡(SE)分析建立了F蛋白TMDs有效地与蛋白质的其余部分分离的三聚体。在多种重要功能突变中发现了TM-TM关联的改变，提示TM-TM相互作用对F蛋白的折叠和功能有调节作用。我们研究的长期目标是 了解副粘病毒F蛋白促进膜融合的分子细节及其调控。这一建议的总体假设是，TMD与自身和脂质环境的相互作用对融合蛋白的稳定性和功能至关重要，使这些相互作用成为抗病毒治疗的潜在靶点。为了验证这个重要的假设， 我们将追求三个具体目标。首先，我们将定义驱动F蛋白TM-TM相互作用的关键因素，包括确定L/I七肽在TM-TM结合和Hendra F蛋白稳定性中的作用。其次，我们将确定TMD如何影响融合的触发和膜融合的促进，重点是C末端β-分支残基的作用，以及当TM-TM相互作用不能释放时可能发生的构象变化。第三，在细胞膜和病毒膜的背景下，我们将剖析脂环境在灌流F稳定性中的作用。这些重要的实验将为TMDs在调节F蛋白稳定性和促进融合中的作用提供关键的新见解。

DESCRIPTION (provided by applicant): Compelling work in the last decade has shown that transmembrane domains (TMDs), once thought to function solely as hydrophobic anchors, are central players driving cellular protein oligomerization, cell signaling, and regulation of channel function. However, the role of TMDs in viral glycoprotein stability and function remains poorly understood. During the previous funding period, our studies of the Hendra virus (HeV) fusion (F) protein identified key roles for TMDs in regulation of F protein stability, cellular trafficking, ad fusion function. Our novel studies utilizing sedimentation equilibrium (SE) analysis established that F protein TMDs efficiently trimerize separate from the rest of the protein. Altered TM-TM association was found for multiple functionally important mutations, implicating TM-TM interactions in regulation of F protein folding and function. The long-term goal of our research is to understand the molecular details and regulation of membrane fusion promoted by the paramyxovirus F proteins. The overall hypothesis of this proposal is that interactions of the TMD with itself and the lipid environment are critical for fusion protein stability and function, makin these interactions potential targets for antiviral therapeutics. To test this important hypothesis, we will pursue three specific aims. First, we will define key elements driving F protein TM-TM interactions including defining the role of a L/I heptad in TM-TM association and Hendra F protein stability. Second, we will determine how the TMD affects fusion triggering and promotion of membrane fusion, with a focus on the role of C-terminal beta-branched residues and a determination of the conformational changes that can occur when TM-TM interactions cannot be released. Third, we will dissect the role of the lipid environment in prefusion F stability in the context of cellular and viral membranes. These important experiments will provide critical new insight into the role of TMDs in regulation of F protein stability and fusion promotion.