Characterizing functionally relevant HIV-1 structures by correlative light and cryo-electron microcopy (CLEM)

通过相关光和冷冻电子显微镜 (CLEM) 表征功能相关的 HIV-1 结构

基本信息

批准号：
10700544
负责人：
Ashwanth Christopher Francis
金额：
$ 19.25万
依托单位：
FLORIDA STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-01-18 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10700544
关键词：
3-Dimensional Architecture Avidity Behavior Calibration Capsid Cell Nucleus Cells Cellular biology Classification Complementary DNA Complex Computer software Cryo-electron tomography Cryoelectron Microscopy Data Data Collection Detection Development Dimensions Dose DsRed Electrons Elements Fluorescence Freezing Genes Genetic Transcription Genome Genomics HIV HIV-1 Ice Image In Situ In Vitro Infection Integrase Integration Host Factors Investigation Ions Label Light Location Macrophage Maps Mediating Methods Nature Nuclear Nuclear Import Nuclear Pore Nuclear Pore Complex PF4 Gene Positioning Attribute Preparation Procedures Productivity Public Health RNA Reporting Reproducibility Resolution Reverse Transcription Sampling Site Specimen Structure System T-Lymphocyte Testing Thinness Tomogram Training Validation Viral Genome Viral Reverse Transcription Virus Virus Integration Virus Replication Visualization cell type experience imaging approach improved insight interest live cell imaging monocyte neural network particle preservation recruit sensor structural imaging success tool trafficking viral DNA

项目摘要

Project Summary The HIV-1 capsid protects the viral genome from host innate sensors and mediates the nuclear import of pre- integration complexes. At some point during intra-cellular trafficking the capsid must disassemble to release the reverse transcribing vDNA for its integration into host-genes. The underlying mechanisms and the cellular location of capsid disassembly, which is commonly referred to as uncoating, remain unclear. Recent evidence points to the possibility to transport a few apparently intact capsids (dimensions ~30 x 60 nm) through a dilated (~60 nm) nuclear pore complex and into the nucleus1-3. The relevance of these few capsids to infection remains unclear, and their prior dynamics inside cells unknown. An unbiased and detailed structural analysis of relevant intra-cellular HIV-1 uncoating structures by cryo-EM remains highly challenging. Notably, in situ cryo-EM necessitates the use of cryo-focused ion beam (cryo-FIB) milling to generate a thin (100-300nm) sheet of vitreous ice referred to as a ‘lamella’ inside rapidly frozen cells. Procedures to precisely control the location in a cell where the lamella is prepared remains underdeveloped and a bottleneck for in situ structural investigation, especially of sparsely located HIV-1 specimen. The scientific premise of this proposal is to develop a robust 3D correlative light and cryo-EM (3D-CLEM) workflow to facilitate cryo-FIB ‘lamella’ preparation at precise intra-cellular sites containing HIV-1, and to characterize relevant capsid structures and correlate to their functional dynamics by live-cell imaging. This project will leverage, (1) fluorescence live-cell imaging of HIV-1 infection to locate relevant capsids that are respectively poised for nuclear entry and integration4, 5 and correlate their dynamics to capsid structures determined by 3D-CLEM, cryo-FIB lamella preparation and high-resolution cryo-electron tomography (cryo-ET); (2) develop automated methods to locate HIV-1 macrostructures on a lamella using low-resolution cryo-ET maps via a convoluted neural network (CNN)-trained software6 to un-biasedly pick capsid templates and 3D-HIV structures on a lamella, for high-resolution cryo-ET structural imaging and classification. The development and validation of the 3D-CLEM workflow and automated macrostructure localization procedure will improve throughput in cryo-EM imaging of HIV-1 structures inside cells, and improve our understanding of virus cell biology, which is of high public health significance.

项目概要 HIV-1衣壳保护病毒基因组免受宿主先天传感器的影响，并介导前病毒的核输入在细胞内运输过程中的某个时刻，衣壳必须分解以释放整合复合物。逆转录 vDNA 整合到宿主基因中的基本机制和细胞。衣壳解体（通常称为脱壳）的位置仍不清楚。指出通过扩张的通道运输一些表面上完整的衣壳（尺寸〜30 x 60 nm）的可能性 (~60 nm) 核孔复合体并进入细胞核1-3 这些衣壳与感染的相关性仍然存在。不清楚，并且它们在细胞内的先前动态未知。对相关的公正且详细的结构分析。值得注意的是，通过冷冻电镜观察细胞内 HIV-1 脱壳结构仍然极具挑战性。需要使用冷冻聚焦离子束 (cryo-FIB) 铣削来生成薄 (100-300 nm) 玻璃体片冰被称为快速冷冻细胞内的“薄片”，用于精确控制细胞中的位置。制备的薄片仍然不发达，是原位结构研究的瓶颈，特别是该提案的科学前提是开发强大的 3D 相关光。和冷冻电镜 (3D-CLEM) 工作流程，以促进冷冻 FIB“薄片”在精确的细胞内位点制备，其中包含 HIV-1，并通过活细胞成像表征相关衣壳结构并与其功能动态相关联。该项目将利用 (1) HIV-1 感染的荧光活细胞成像来定位相关衣壳分别准备好进入核和整合4、5，并将它们的动态与确定的衣壳结构相关联通过 3D-CLEM、冷冻 FIB 薄片制备和高分辨率冷冻电子断层扫描 (cryo-ET) (2) 开发；使用低分辨率冷冻 ET 图通过自动化方法定位薄片上的 HIV-1 宏观结构经过卷积神经网络 (CNN) 训练的软件6，可以无偏见地挑选衣壳模板和 3D-HIV 结构薄片，用于高分辨率冷冻电子断层扫描结构成像和分类。 3D-CLEM 工作流程和自动化宏观结构定位程序将提高冷冻电镜的通量 HIV-1细胞内结构的成像，提高我们对病毒细胞生物学的理解，这具有很高的价值公共卫生意义。