Dissecting the Conformational Flexibility of HIV-1 Envelope Glycoproteins

摘要 HIV-1包膜糖蛋白(Env)介导病毒进入宿主细胞，是唯一的中和靶点。 抗体。大多数主要HIV-1毒株的HIV-1包膜病毒通常以封闭构象存在(状态1)和 偶尔过渡到下游更开放的构象(状态2和状态3)。因此，目前的知识 引导免疫原设计模拟Env闭合构象作为诱导的首选目标 广谱中和抗体(BNAbs)，以阻止HIV-1进入。然而，环境更喜欢更多的构象 3700多万种不同的HIV-1循环毒株不太可能采用完全相同的封闭构象。和 我们仍然不知道不同的天然艾滋病毒-1变种的国家1构象有多大的不同。 我们的假设：状态1环境形成了一组异质环境，它们存在于相似但不相同的环境中 构象。因此，改进的bNAbs识别多个环境构象的能力将 扩大它们的中和活性。 为了验证我们的假设，我们将评估来自不同菌株的env的构象异质性，并建立 1)环境转换的机制模型，2)存在于特定环境中的环境的生成和传输 构象；3)对不同环境状态的广泛识别。在具体目标1中，我们将研究构象 传播/创始(T/F)HIV-1毒株的多样性，这可以在体内建立HIV-1，是唯一的目标 一种预防性疫苗。我们将研究T/F环境暴露内部元素的机制以及它们是如何 过渡到下游构象。在具体目标2中，我们将研究 不完全封闭的环境构象。我们将重建T/F在患者体内的演变路径 在不同时间点构建进化病毒的可用病毒序列，然后调查 随着时间的推移它们的构象状态。同时，我们将研究HIV-1毒株之间的环境构象多样性 存档在HIV-1携带者的潜在HIV-1储存库中，并确定潜在的目标 治疗性干预。这些方法将使我们能够建立一个机械模型，说明 封闭的环境被生成、演化和传输。在具体目标3中，我们将为 广泛识别不同原代毒株不同环境构象上的CD4结合位点(CD4-bs)。我们 将研究异常广泛的N6(CD4-bs)bNab如何识别不同的环境构象，将识别 与开放和封闭环境构象上残基的临界相互作用，并将解决 HIV-1的包膜蛋白与N6形成复合体，定义了广泛识别的机制。 总体而言，我们的研究将为HIV-1进入途径的生物学提供高分辨率和全面的观点， 将完善关于HIV-1环境构象的知识，并将为开发新的 HIV-1治疗和疫苗设计的策略。

ABSTRACT HIV-1 envelope glycoproteins (Envs) mediate viral entry into host cells and are the sole target of neutralizing antibodies. HIV-1 Envs of most primary HIV-1 strains typically exist in a closed conformation (State 1) and occasionally transit to downstream, more open conformations (State 2 and State 3). Thus, current knowledge guides immunogen design towards mimicking the Env closed conformation as the preferred target for eliciting broadly neutralizing antibodies (bnAbs) to block HIV-1 entry. However, Env preferred conformations of more than 37 million different circulating HIV-1 strains are unlikely to adopt exactly the same closed conformation. And we still do not know how different State 1 conformations of diverse natural HIV-1 variants are. Our hypothesis: State 1 Envs form a heterogenous group of Envs that exist in similar but not identical conformations. Accordingly, improved ability of bnAbs to recognize multiple Env conformations will broaden their neutralization activity. To test our hypothesis, we will assess the conformational heterogeneity of Envs from different strains and build mechanistic models for 1) Envs transitions, 2) generation and transmission of Envs that exist in specific conformations, and 3) broad recognition of different Env states. In Specific Aim 1 we will study the conformational diversity of transmitted/founder (T/F) HIV-1 strains, which can establish HIV-1 in vivo and are the only target of a preventive vaccine. We will study the mechanisms by which T/F Envs expose internal elements and how they transit to downstream conformations. In Specific Aim 2 we will investigate the generation and evolution of incompletely closed Env conformation. We will reconstruct the evolution pathway of T/Fs in patients by using available viral sequences to build evolved viruses at different time point followed by investigating changes in their conformational states over time. In parallel, we will study Env conformational diversity among HIV-1 strains that are archived in the latent HIV-1 reservoir of people living with HIV-1 and identify potential targets for therapeutic interventions. These approaches will allow us to build a mechanistic model for how incompletely closed Envs are generated, evolved, and transmitted. In Specific Aim 3 we will define important parameters for broad recognition of CD4-binding site (CD4-bs) on different Env conformations of diverse primary strains. We will study how the exceptionally broad N6 (CD4-bs) bnAb recognizes different Env conformations, will identify critical interactions with residues on open and closed Env conformations, and will solve cryo-EM structures of HIV-1 Envs in complex with N6 to define the mechanism of broad recognition. Overall, our study will provide high-resolution and comprehensive view on the biology of HIV-1 entry pathway, will refine the knowledge on HIV-1 Env conformations, and will form a strong basis for the development of new strategies for HIV-1 cure and vaccine design.