Single molecule imaging of HIV-1 entry

 描述（由申请人提供）：HIV-1包膜蛋白（Env）是介导病毒进入细胞的1类膜融合机器。HIV-1 Env由gp 120/gp 41异源二聚体的三聚体组成。与CD 4受体的相互作用导致gp 120的结构重排，从而形成辅助受体结合位点。随后与辅助受体的相互作用触发额外的Env重折叠，gp 41重排成稳定的六螺旋束，据信驱动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三聚体融合前构象和gp 41融合后构象的静态图像。然而，直接可视化的构象转变，表征的结构中间体，导致膜融合一直缺乏。为了深入了解天然三聚体的动力学，我们建立了单分子荧光共振能量转移（smFRET）成像来测量单个Env分子在完整病毒体表面上的天然三聚体背景下的构象变化。我们的研究表明，unliganded HIV-1 Env本质上是动态的，在三种不同的融合前构象之间转换，其相对位置由受体CD 4和抗体结合重塑。我们的分析还直接揭示了gp 120中的分子和时间事件，这些事件是通过必要的结构中间体由CD 4和辅助受体两步激活HIV-1 Env的基础。在这里，我们建议集中在下一个重要的目标，直接可视化的构象事件内的gp 41，导致病毒和细胞膜之间的混合。为此，我们已经建立了抑制tRNA技术，在哺乳动物细胞中引入非天然氨基酸。在gp 41中引入单个荧光团与gp 120中现有的荧光团组合将监测CD 4如何引发gp 41和共受体的结合破坏gp 41和gp 120之间的缔合。在gp 41的HR 1和HR 2区域内的开关区域之前和之后插入两个荧光团将揭示Env三聚体内的个体构象转变如何导致融合肽暴露和膜融合。因为我们的方法允许洞察天然HIV-1 Env三聚体在完整病毒体表面上的构象状态，所以我们还独特地定位于评估目前用于结构表征HIV-1 Env三聚体的可溶性或前体构建体在多大程度上展示天然Env的特征。对HIV-1 Env激活的基础构象事件的进一步了解将为抗病毒治疗和疫苗设计提供信息。

 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.