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

• 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
• 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

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.

项目总结