Structure and Function of Viral Fusion Domains

包膜病毒与其靶细胞的融合是由病毒跨膜蛋白介导的。 糖蛋白这种蛋白质与细胞膜相互作用的第一部分称为融合结构域，并且是通常靠近氨基末端的保守的、大部分疏水的多态性序列。定点诱变已经显示，流感病毒血凝素（HA 2）或HIV糖蛋白41，000（gp 41）的融合结构域中的关键残基的替换影响病毒融合。与这些N-末端区域具有相同序列的合成肽（称为融合肽（FP））诱导脂质混合以及脂质体和细胞膜的裂解。虽然有很多关于病毒FP结构的信息，但对膜内FP结构与其功能的关系了解甚少。拟议研究的主要目的是确定融合能力所必需的病毒FP的共同结构特征。使用基于突变病毒序列的FP，我们将 寻找它们改变的活性和膜结合结构之间的相关性。我们还将进一步表征C-螺旋（DP-178）或其片段对FP的抑制作用，并评估FP扩展其构象空间以包括淀粉样蛋白超结构形成的能力。FP及其变体的膜扰动活性将通过红细胞溶解和聚集（通过540 nm处释放的血红蛋白的吸光度测量）以及使用Coulter计数器的细胞大小进行筛选。将使用荧光去猝灭和光散射测定来测量FP诱导的合成大单层囊泡的脂质混合、泄漏和聚集。用圆二色谱法检测融合肽在膜或膜模拟溶剂中的构象、取向和形貌 (CD)傅里叶变换红外（FTIR）、电子自旋共振（ESR）光谱和分子模拟。将寻求结构模型与病毒融合肽和变体诱导的融合活性和脂质扰动之间的相关性。

The fusion of enveloped viruses with their target cells is directed by the viral transmembrane glycoprotein. The first part of this protein to interact with the cellular membrane is called the fusion domain and is a conserved, largely hydrophobic, polymorphic sequence usually near the amino terminus. Site-directed mutagenesis has shown that the replacement of key residues in the fusion domain of influenza virus hemagglutinin (HA2) or HIV glycoprotein 41,000 (gp41) affect viral fusion. Synthetic peptides with the same sequences as these N-terminal regions, termed fusion peptides (FP), induce lipid mixing and lysis of liposomes and cell membranes. Although there is much information on the structure of viral FP, there is little understanding of the relationship of the intramembrane FP structures to their function. The principal objective of the proposed research is to determine the structural characteristics common to viral FP that are necessary for fusion competence. Using FP based on mutated viral sequences, we will seek correlations between their altered activity and membrane-bound structures. We will also further characterize the inhibition of FP by the C-helix (DP-178) or its fragments and we will assess the ability of FP to expand its conformational space to include the formation of amyloid suprastructures. Membrane-perturbing activities of FP and its variants will be screened with erythrocyte lysis and aggregation measured by the absorbance of released hemoglobin at 540nm and cell sizing with a Coulter Counter. Lipid mixing, leakage, and aggregation of synthetic large unilamellar vesicles induced by FP will be measured using fluorescence dequenching and light scattering assays. The conformation, orientation, and topography of fusion peptides in membranes or membrane mimmicking solvents will be examined by circular dichroism (CD), Fourier transform infrared (FTIR), electron spin resonance (ESR) spectroscopy and molecular modeling. Correlations will be sought between the structural models and the fusion activities and lipid perturbations induced by viral fusion peptides and variants.