Single molecule imaging of HIV-1 entry

 描述(申请人提供)：HIV-1包膜蛋白(Env)是一种1类膜融合机，介导病毒进入细胞。HIV-1Env由gp120/gp41杂二聚体三聚体组成。与CD4受体的相互作用导致gp120的结构重排，这导致形成一个辅助受体结合部位。随后与辅助受体的相互作用引发额外的Env重折叠，gp41重排成稳定的六螺旋束，被认为推动病毒和细胞膜之间的融合。最近在结构理解方面的进展提供了HIV-1 Env三聚体融合前构象和gp41融合后构象的静态图像。然而，导致膜融合的构象转变的直接可视化和结构中间体的表征一直是缺乏的。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)成像技术，以测量单个Env分子在完整病毒粒子表面天然三聚体背景下的构象变化。我们的研究表明，去连接的HIV-1Env是内在的动态的，在三种不同的预融合构象之间转换，其相对占有率被受体CD4和抗体结合所重塑。我们的分析还直接揭示了gp120中的分子和时间事件，这些事件是CD4和辅助受体通过必要的结构中介两步激活HIV-1Env的基础。在这里，我们建议专注于下一个重要的目标，直接可视化gp41内导致病毒和细胞膜混合的构象事件。为此，我们建立了抑制tRNA技术，在哺乳动物细胞中引入非天然氨基酸。在gp41中引入一个单一的荧光团，与gp120中现有的一个荧光团相结合，将监测CD4Primes gp41和辅助受体的结合如何破坏gp41和gp120之间的联系。在gp41的HR1和HR2区域的切换区之前和之后插入两个荧光团将揭示环境三聚体内的单个构象转变如何导致融合肽暴露和膜融合。由于我们的方法允许深入了解天然HIV-1 Env三聚体在完整病毒粒子表面的构象状态，我们也具有独特的优势，可以评估目前用于表征HIV-1 Env三聚体结构特征的可溶性或前体结构。对HIV-1Env激活的构象事件的更多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.