Structures of initial CD4 engagement with pre-fusion, closed HIV-1 Envelope trimer and early CD4-induced conformational changes required for infection

在融合前，封闭的HIV-1包膜(Env)三聚体上的CD4受体的初始接触位置是高度 保守，并且是中和抗体的靶标。该站点是疫苗和疫苗的重要目标 治疗学的发展。这种初始交互的结构定义一直是一个挑战，因为 HIV-1环境三聚体的构象变化紧随其后的是CD4结合。没有很高的 融合前的初始接触的解析结构，闭合的HIV-1环境三聚体与CD4。结构性的 CD4结合后立即发生的构象变化的细节也是未知的。精确 定义与封闭的HIV-1环境三聚体的初始CD4接触将填补我们对这一早期理解的一个空白 事件，并将为基于结构的免疫原设计提供原子水平信息。这个 因此，这项研究的总体目标是：(I)在原子水平上详细定义CD4结合的初始位置 融合前，封闭的HIV-1环境三聚体，(Ii)定义导致CD4诱导的原核糖体开放的步骤，以及(Iii) 为了阐明CD4介导的HIV-1融合肽的变化，HIV-1融合肽是关键的免疫优势区域 介导HIV-1CD4T细胞进入，本身也是中和抗体的靶标。这其中的科学前提是 格兰特认为，CD4与HIV-1环境病毒的初始接触是决定病毒附着的关键第一步。 尽管cd4可以与多种env构象结合，但HIV-1env三聚体上的这个第一个接触部位是 广泛中和抗体和有效药物的靶标，因此高分辨率的结构细节 相互作用和对随后构象变化的机械性理解将促进 制定干预策略，包括针对HIV-1疫苗和药物的免疫原设计 设计新的治疗艾滋病的策略，以消除潜伏池的HIV-1感染的CD4T细胞。这个 这笔赠款的创新来自低温电磁技术的进步，其中包括新的栅格准备和 标本玻璃化方法、改进的显微镜硬件、高通量数据的自动化方法 集合，以及用于数据处理的高级算法。这些进步最近使我们能够 建立快速测定HIV-1包膜复合体高分辨结构的方法。创新 也源于可获得提议的结构的原生类环境构建物和抗体的面板 和机械论分析。在这项研究完成后，我们预计将提供一部关于CD4诱导的电影 打开HIV-1环境病毒三聚体。HIV-1封闭环境中初始接触的高分辨结构 Trimer将为基于结构的免疫原和药物设计提供原子级信息。可视化 CD4诱导的Env开放的初始步骤将提供关于哪些Env区域最先移动的信息，并将 告知稳定免疫原的设计。这项研究也将提供对CD4诱导的 在HIV-1融合多肽区域采样的构象多样性以及天然和 疫苗引发的，对它有反应。

The initial contact site of the CD4 receptor on pre-fusion, closed HIV-1 envelope (Env) trimer is highly conserved, and is targeted by neutralizing antibodies. This site is an important target for vaccine and therapeutics development. Structural definition of this initial interaction has been a challenge because conformational changes in the HIV-1 Env trimer follow immediately upon CD4 engagement. There are no high resolution structures of the initial contact of the pre-fusion, closed HIV-1 Env trimer with CD4. The structural details of the conformational changes that follow immediately upon CD4 binding are also not known. Precise definition of initial CD4 contacts with the closed HIV-1 Env trimer will fill a gap in our understanding of this early event in HIV-1 entry, and will provide atomic level information for structure-based immunogen design. The overall goals of this study, therefore are, (i) to define, at atomic level details, the initial site of CD4 binding on pre-fusion, closed HIV-1 Env trimer, (ii) to define the steps leading to CD4-induced protomer opening, and (iii) to elucidate CD4-mediated changes in the HIV-1 fusion peptide, an immunodominant region critical for mediating HIV-1 CD4+ T cell entry, and itself a target of neutralizing antibodies. The scientific premise of this grant is that the initial contact of CD4 with HIV-1 Env is the critical first step that determines virus attachment. Although CD4 can bind to multiple Env conformations, this first site of contact on the HIV-1 Env trimer is the target of broadly neutralizing antibodies and effective drugs, hence high resolution structural details of this interaction and a mechanistic understanding of subsequent conformational changes will facilitate the development of intervention strategies that include immunogen design for HIV-1 vaccine efforts and drug design for novel cure AIDS strategies to eliminate the latent pool of HIV-1-infected CD4 T cells. The innovation in this grant derives from advances in cryo-EM technology that include new grid preparation and specimen vitrification methods, improved microscope hardware, automated methods for high-throughput data collection, and advanced algorithms for data processing. These advances have recently allowed us to establish a rapid pipeline for determining high resolution structures of HIV-1 Env complexes. The innovation also derives from availability of panels of native-like Env constructs and antibodies for the proposed structural and mechanistic analyses. At the completion of this study we expect to provide a movie for CD4-induced opening of the HIV-1 Env trimer. High resolution structures of the initial CD4 contact on the closed HIV-1 Env trimer will provide atomic level information for structure-based immunogen and drug design. Visualization of the initial steps of CD4-induced Env opening will provide information on which Env regions move first, and will inform the design of stabilized immunogens. This study will also provide an understanding of the CD4-induced conformational diversity sampled at the HIV-1 fusion peptide region and how antibodies, both natural and vaccine-elicited, respond to it.