Solid-State NMR of Viral Fusion Peptides and Proteins

项目总监/主要研究者（最后一位、第一位、中间一位）：Weliky，大卫保罗 项目总结/摘要 拟议研究的长期目标是详细了解病毒融合蛋白的基础- 诱导膜融合。病毒和靶细胞膜的融合是许多病毒感染的关键步骤 重要的疾病和融合机制的详细了解将有助于抗病毒药物的开发， 其作用方式是融合抑制的治疗剂。融合蛋白也是病毒疫苗的靶点 发展本研究的重点是研究血凝素HA 2和gp 41的融合蛋白。 流感病毒和人类免疫缺陷病毒。选择这些蛋白质是因为 病毒引起主要的人类疾病，并且因为这些蛋白质充当其他I类病毒融合的原型 proteins.虽然蛋白质序列是非同源的，但两种蛋白质都是单程跨膜的 病毒膜蛋白，具有约180个残基的胞外域区域，位于病毒外部。有 特别强调在融合中起关键作用的~25个残基的N-末端“融合肽”（FP）结构域 催化作用，并被认为在融合过程的早期插入宿主细胞膜。有四个目标 包括HA 2（Aim 1）和gp 41（Aim 2）的FP在膜中的结构分布，以及 HA 2（目标3）和gp 41（目标4）的FP的膜位置。每个目标分为（a）仅FP和（B） FP+可溶性胞外域+跨膜结构域切片。两种蛋白质的FP都可以采用α螺旋 或反平行β折叠结构，并且这两种结构都催化膜融合。该项目的重点是α HA 2和β gp 41 FP结构。结构/功能相关性进一步发展与推论 对HA 2 G1 E和gp 41 V2 E突变体的测量结果表明，这两种突变体均导致融合和病毒感染大大受损。 感染阐明FP结构和膜接触的主要技术是固态核磁共振 特别强调旋转回波双共振（REDOR），可以阐明 核间接近度和距离≤ 10 Å。REDOR将用于确定螺旋间HA 2 FP结构 和β gp 41 FP结构的反平行登记。将确定不同的部分人群 结构以及结构和群体如何随着融合损害突变而变化。会有 FP/膜接触的REDOR测量以及FP/膜接触的互补SSNMR测量 使用顺磁弛豫和1H自旋扩散的FP/水接触。 OMB编号0925-0001/0002（2016年3月修订版，批准至2018年10月31日）

Program Director/Principal Investigator (Last, First, Middle): Weliky, David Paul Project Summary/Abstract The long-term goal of the proposed research is detailed understanding of the basis for viral fusion protein- induced membrane fusion. Fusion of viral and target cell membranes is a key step in infection for many viruses important in disease and a detailed understanding of fusion mechanisms will aid development of anti-viral therapeutics whose mode of action is fusion inhibition. Fusion proteins are also a target of viral vaccine development. The proposed research focuses on the hemagglutinin HA2 and gp41 fusion proteins of the influenza virus and human immunodeficiency virus, respectively. These proteins are chosen because these viruses cause major human diseases and because the proteins serve as prototypes for other class I viral fusion proteins. Although the protein sequences are non-homologous, both proteins are single-pass transmembrane proteins of the viral membrane with ~180-residue ectodomain regions which lie outside the virus. There is particular emphasis on the ~25-residue N-terminal “fusion peptide” (FP) domain which plays a key role in fusion catalysis and is thought to insert into the host cell membrane early in the fusion process. There are four Aims which include structural distributions in membrane of the FP's of HA2(Aim 1), and gp41(Aim 2), and the membrane locations of the FP's of HA2(Aim 3) and gp41(Aim 4). Each Aim is divided into (a) FP-only and (b) FP+soluble ectodomain+transmembrane domain sections. The FP's of both proteins can adopt either α helical or antiparallel β sheet structure, and both structures catalyze membrane fusion. The project focuses on the α HA2 and β gp41 FP structures. Structure/function correlations are further developed with corollary measurements on HA2 G1E and gp41 V2E mutants which both result in greatly impaired fusion and viral infection. The main technique to elucidate FP structure and membrane contacts is solid-state nuclear magnetic resonance(SSNMR) with particular emphasis on rotational-echo double resonance(REDOR) which can elucidate internuclear proximities and distances ≤10Å. REDOR will be used to determine interhelical HA2 FP structures and antiparallel registries of β gp41 FP structures. Fractional populations will be determined for the different structures as well as how the structures and populations change with fusion-impairing mutations. There will be REDOR measurements of FP/membrane contacts as well as complementary SSNMR measurements of FP/water contacts using paramagnetic relaxation and 1H spin diffusion. OMB No. 0925-0001/0002 (Rev. 03/16 Approved Through 10/31/2018) Page Continuation Format Page