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

初始 CD4 与融合前接合的结构、封闭的 HIV-1 包膜三聚体以及感染所需的早期 CD4 诱导的构象变化

基本信息

批准号：
10552588
负责人：
Priyamvada Acharya
金额：
$ 77.08万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-02-01 至 2025-01-31
项目状态：
未结题

项目摘要

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.

融合前封闭的 HIV-1 包膜 (Env) 三聚体上 CD4 受体的初始接触位点高度保守，并被中和抗体靶向。该位点是疫苗和疫苗的重要目标治疗学的发展。这种初始相互作用的结构定义一直是一个挑战，因为 HIV-1 Env 三聚体的构象变化在 CD4 结合后立即发生。没有高的融合前、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详细信息也是未知的。精确的 CD4 与封闭的 HIV-1 包膜三聚体的初始接触的定义将填补我们对这一早期接触的理解空白。 HIV-1进入中的事件，将为基于结构的免疫原设计提供原子级信息。这因此，本研究的总体目标是，(i) 在原子水平细节上定义 CD4 结合的初始位点。融合前，闭合 HIV-1 Env 三聚体，(ii) 定义导致 CD4 诱导的原聚体开放的步骤，以及 (iii) 阐明 HIV-1 融合肽中 CD4 介导的变化，这是免疫显性区域，对于介导 HIV-1 CD4+ T 细胞进入，其本身也是中和抗体的目标。这样做的科学前提授予的观点是，CD4 与 HIV-1 包膜的初次接触是决定病毒附着的关键的第一步。尽管 CD4 可以结合多个 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标，因此具有高分辨率的结构细节相互作用和对随后构象变化的机械理解将有助于制定干预策略，包括 HIV-1 疫苗工作和药物的免疫原设计设计新的艾滋病治疗策略，以消除潜在的 HIV-1 感染的 CD4 T 细胞池。这这笔赠款的创新源于冷冻电镜技术的进步，包括新的网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一个快速管道来确定 HIV-1 Env 复合物的高分辨率结构。创新还源于针对拟议结构的天然样 Env 构建体和抗体组的可用性和机理分析。在这项研究完成后，我们希望为 CD4 诱导提供一部影片 HIV-1 Env 三聚体的打开。封闭 HIV-1 包膜上初始 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级信息。可视化 CD4 诱导的 Env 打开的初始步骤将提供有关哪些 Env 区域首先移动的信息，并将为稳定免疫原的设计提供信息。这项研究还将提供对 CD4 诱导的 HIV-1 融合肽区域的构象多样性以及天然和抗体如何疫苗引起的，对其做出反应。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Structures of Langya Virus Fusion Protein Ectodomain in Pre- and Postfusion Conformation.

狼牙病毒融合蛋白胞外域融合前和融合后构象的结构。

DOI：
10.1128/jvi.00433-23
发表时间：
2023
期刊：
Journal of virology
影响因子：
5.4
作者：
May,AaronJ;Pothula,KarunakarReddy;Janowska,Katarzyna;Acharya,Priyamvada
通讯作者：
Acharya,Priyamvada

Transient transfection and purification of SARS-CoV-2 spike protein from mammalian cells.

DOI：
10.1016/j.xpro.2022.101603
发表时间：
2022-09-16
期刊：
STAR PROTOCOLS
影响因子：
0
作者：
Stalls, Victoria;Janowska, Katarzyna;Acharya, Priyamvada
通讯作者：
Acharya, Priyamvada

Cold sensitivity of the SARS-CoV-2 spike ectodomain.

DOI：
10.1038/s41594-020-00547-5
发表时间：
2021-03
期刊：
Nature structural & molecular biology
影响因子：
16.8
作者：
Edwards RJ;Mansouri K;Stalls V;Manne K;Watts B;Parks R;Janowska K;Gobeil SMC;Kopp M;Li D;Lu X;Mu Z;Deyton M;Oguin TH 3rd;Sprenz J;Williams W;Saunders KO;Montefiori D;Sempowski GD;Henderson R;Munir Alam S;Haynes BF;Acharya P
通讯作者：
Acharya P