Dissecting the Conformational Flexibility of HIV-1 Envelope Glycoproteins

摘要 HIV-1包膜糖蛋白（Envs）介导病毒进入宿主细胞，是中和HIV-1的唯一靶点。 抗体的大多数原代HIV-1毒株的HIV-1 Env通常以闭合构象（状态1）存在， 偶尔过渡到下游，更开放的构象（状态2和状态3）。目前的知识 引导免疫原设计朝向模拟Env闭合构象作为用于引发免疫应答的优选靶标。 广泛中和抗体（bnAb），以阻止HIV-1进入。然而，Env更喜欢构象 超过3700万种不同的HIV-1病毒株不太可能采用完全相同的闭合构象。和 我们仍然不知道不同的天然HIV-1变体的状态1构象有多大的不同。 我们的假设：状态1 Envs形成了一个异质的Envs组，这些Envs存在于相似但不相同的环境中。 构象因此，bnAb识别多种Env构象的能力的提高将有助于 扩大其中和活性。 为了验证我们的假设，我们将评估来自不同菌株的Envs的构象异质性，并构建 1）Envs转换的机制模型，2）存在于特定环境中的Envs的产生和传播 构象，和3）不同的Env状态的广泛识别。在具体目标1中，我们将研究 传播/创始者（T/F）HIV-1毒株的多样性，这些毒株可以在体内建立HIV-1，并且是HIV-1的唯一靶点。 预防性疫苗我们将研究T/F Envs暴露内部元素的机制，以及它们如何 转移到下游构象。在具体目标2中，我们将研究 不完全闭合的Env构象。我们将通过使用免疫组织化学方法重建患者T/Fs的演变途径， 在不同的时间点构建进化的病毒，然后研究 它们的构象状态。同时，我们将研究HIV-1毒株之间的Env构象多样性 在HIV-1感染者的潜伏HIV-1储存库中存档，并确定潜在的目标， 治疗干预。这些方法将使我们能够建立一个机械模型， 封闭的Env被生成、演化和传输。在具体目标3中，我们将定义重要参数， 广泛识别不同原代菌株的不同Env构象上的CD 4结合位点（CD 4-bs）。我们 将研究异常广泛的N6（CD 4-bs）bnAb如何识别不同的Env构象， 关键的相互作用与残基开放和封闭的Env构象，并将解决冷冻EM结构的 HIV-1 Envs与N6复合以定义广泛识别的机制。 总的来说，我们的研究将提供关于HIV-1进入途径生物学的高分辨率和全面的观点， 将完善关于HIV-1 Env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.