Single molecule imaging of HIV-1 entry

HIV-1 进入的单分子成像

• 批准号: 9321118
• 负责人: WALTHER H MOTHES
• 金额: $ 37.93万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9321118
• 关键词: Amino Acids; Antibodies; Antiviral Therapy; Binding; Binding Sites; CD4 Antigens; Cells; Cellular Membrane; Comparative Study; Cytoplasmic Tail; Dyes; Enzymes; Event; Exhibits; Fluorescence Resonance Energy Transfer; Goals; Grant; HIV; HIV Envelope Protein gp120; HIV-1; Image; Imagery; Individual; Label; Lead; Mammalian Cell; Measurement; Measures; Mediating; Membrane Fusion; Methods; Molecular; Molecular Conformation; Monitor; Peptides; Positioning Attribute; Structure; Surface; Technology; Time; Transfer RNA; Vaccine Design; Viral; Viral Proteins; Virion; Virus; conformational conversion; env Gene Products; fluorophore; insight; molecular imaging; new technology; physical property; protein activation; public health relevance; receptor; single molecule; single-molecule FRET; therapy design; unnatural amino acids

 DESCRIPTION (provided by applicant): The HIV-1 envelope protein (Env) is a class 1 membrane fusion machine that mediates virus entry into cells. HIV-1 Env consists of a trimer of gp120/gp41 heterodimers. Interaction with the CD4 receptor causes structural rearrangements in gp120, which lead to formation of a coreceptor-binding site. Subsequent interactions with the coreceptor trigger additional Env refolding, with gp41 rearranging into a stable six-helix bundle that is believed to drive fusion between viral and cellular membranes. Recent progress in the structural understanding have provided static images of the pre-fusion conformation of the HIV-1 Env trimer and post-fusion conformation of gp41. However, direct visualization of the conformational transitions, characterization of structural intermediates that lead to membrane fusion have been lacking. To provide insights into the dynamics of the native trimer, we have established single molecule fluorescence resonance energy transfer (smFRET) imaging to measure the conformational changes of individual Env molecule in the context of a native trimer on the surface of intact virions. Our studies revealed that the unliganded HIV-1 Env is intrinsically dynamic, transitioning between three distinct prefusion conformations, whose relative occupancies were remodeled by receptor CD4 and antibody binding. Our analysis also directly reveals molecular and temporal events in gp120 that underlie the two-step activation of HIV-1 Env by CD4 and coreceptor through a necessary structural intermediate. Here we propose to concentrate on the next important goal, to directly visualize the conformational events within gp41 that lead to mixing between viral and cellular membranes. Towards this end, we have established suppressor tRNA technologies to introduce unnatural amino acids in mammalian cells. The introduction of a single fluorophore in gp41 in combination with an existing one in gp120 will monitor how CD4 primes gp41 and binding of co-receptor disrupts the association between gp41 and gp120. The insertion of two fluorophores before and after switch regions within HR1 and HR2 regions of gp41 will reveal how individual conformational transitions within the Env trimer lead to fusion peptide exposure and membrane fusion. Because our methods allows insights into the conformational state of native HIV-1 Env trimer on the surface of complete virions, we are also uniquely positioned to evaluate to what extent soluble or precursor constructs currently used to structurally characterize the HIV-1 Env trimer display features of the native Env. An increased understanding of the conformational events underlying HIV-1 Env activation will inform antiviral therapies and vaccine design.

 描述（由适用提供）：HIV-1 Invelope蛋白（ENV）是一类膜融合机，可介导病毒进入细胞。 HIV-1 ENV由GP120/GP41异二聚体的触发器组成。与CD4受体的相互作用会导致GP120的结构重排，从而导致形成共受体结合位点。随后与共感染者的相互作用触发了其他ENV进行了重新折叠，GP41重新排列为一个稳定的六螺旋束，据信可以促进病毒和细胞膜之间的融合。结构理解的最新进展提供了HIV-1 ENV三聚体和GP41融合后构象的融合前构象的静态图像。但是，缺乏构象转变的直接可视化，导致膜融合的结构中间体的表征。为了洞悉天然三聚体的动力学，我们已经建立了单分子荧光能传递（SMFRET）成像，以在完整病毒表面的天然三聚体的背景下测量单个ENV分子的概念变化。我们的研究表明，非配合的HIV-1 Env是本质动态的，在三种不同的预融合构象之间过渡，其相对占用是通过受体CD4和抗体结合重塑的。我们的分析还直接揭示了GP120中的分子和临时事件，这些事件是通过必要的结构中间体通过CD4和CORECEPTOR对HIV-1 ENV的两步激活的基础。在这里，我们建议专注于下一个重要目标，直接可视化GP41中导致病毒和细胞膜之间混合的会议事件。为此，我们已经建立了抑制器tRNA技术，以在哺乳动物细胞中引入不自然的氨基酸。 GP41中的单个荧光团与GP120中现有的荧光团的引入将监测CD4 Primes GP41和共受体的结合如何破坏GP41和GP120之间的关联。 GP41的HR1和HR2区域内和之后的两个荧光团的插入将揭示ENV触发内的单个构象转变如何导致融合肽的暴露和膜融合。由于我们的方法允许对完整病毒表面上天然HIV-1 env触发的构象状态进行深入了解，因此我们也具有唯一的定位，以评估目前用于在结构上表征HIV-1 Envigger触发触发触发显示特征的固体或前体构造的程度。对HIV-1环境激活基础的会议事件的越来越多的了解将为抗病毒疗法和疫苗设计提供信息。