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

CD4受体在融合前封闭的HIV-1包膜（Env）三聚体上的初始接触位点高度依赖于CD4受体。 保守，并被中和抗体靶向。该位点是疫苗的重要靶点， 治疗学发展。这种初始相互作用的结构定义一直是一个挑战， HIV-1Env三聚体的构象变化在CD4接合后立即发生。没有高 融合前封闭的HIV-1 Env三聚体与CD4的初始接触的解析结构。结构性 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 Env三聚体的初始CD4接触将填补我们对这一早期认识的空白。 事件的HIV-1进入，并将提供原子水平的信息，基于结构的免疫原设计。的 因此，本研究的总体目标是：（i）在原子水平上详细定义CD4结合的初始位点， 融合前，封闭的HIV-1 Env三聚体，（ii）定义导致CD4诱导的原聚体开放的步骤，和（iii） 为了阐明HIV-1融合肽中CD4介导的变化， 介导HIV-1 CD 4 + T细胞进入，本身就是中和抗体的靶点。科学的前提是 格兰特认为，CD4与HIV-1 Env的最初接触是决定病毒附着的关键第一步。 虽然CD4可以结合多种Env构象，但HIV-1 Env三聚体上的第一个接触位点是 广泛中和抗体和有效药物的靶点，因此该靶点的高分辨率结构细节 相互作用和随后的构象变化的机械理解将有助于 制定干预策略，包括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HIV-1感染的CD4 T细胞的潜伏池。的 这项资助的创新来自于冷冻EM技术的进步，包括新的网格制备， 样品玻璃化方法，改进的显微镜硬件，高通量数据的自动化方法 收集和先进的数据处理算法。这些进步最近使我们能够 建立了一条快速测定HIV-1 Env复合物高分辨率结构的管道。创新 也源于天然样Env构建体和抗体组的可用性，用于所提出的结构 机械分析。在这项研究完成后，我们希望提供一个电影的CD4诱导 打开HIV-1 Env三聚体。封闭的HIV-1 Env上初始CD4接触的高分辨率结构 三聚体将为基于结构的免疫原和药物设计提供原子水平的信息。可视化 CD4诱导的Env开放的初始步骤将提供关于哪个Env区域首先移动的信息，并且将 为设计稳定的免疫原提供了信息。这项研究还将提供对CD4诱导的 在HIV-1融合肽区域取样的构象多样性以及天然和 疫苗引发的反应

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.