CHEETAH Center for the Structural Biology of HIV Infection, Restriction, and Viral Dynamics

CHEETAH HIV 感染、限制和病毒动力学结构生物学中心

• 批准号: 10663363
• 负责人: Pamela J Bjorkman
• 金额: $ 170.74万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-11 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10663363
• 关键词: 3-Dimensional; Architecture; Binding; Biological Assay; Biology of HIV Infection; Capsid; Cell Communication; Cell Nucleus; Cell membrane; Cell-Free System; Cells; Chromatin; Complementary DNA; Complex; Coupled; DNA; DNA Integration; Development; Docking; Electrons; Exposure to; Fluorescence Microscopy; Foundations; Genome; HIV-1; Host Factor 1 Protein; Image; In Situ; Infection; Integration Host Factors; Knowledge; Learning; Life Cycle Stages; Macaca; Methods; Molecular; Molecular Conformation; Nuclear; Nuclear Import; Nuclear Pore; Nucleosome Core Particle; Pathway interactions; Plastic Embedding; Process; Productivity; Proviruses; RNA; Replication Initiation; Resolution; Reverse Transcription; Role; Series; Stains; Structure; System; Systems Integration; Time; Tissue Sample; Transcription Initiation; Ubiquitin; Viral; Viral Reverse Transcription; Virus; Virus Integration; Visualization; design; dimer; electron tomography; experimental study; genomic RNA; in vivo; inhibitor; novel; receptor; reconstitution; simian human immunodeficiency virus; structural biology; transcriptional coactivator p75; viral DNA; viral RNA; viral genomics

PROJECT SUMMARY To initiate an infection, HIV-1 must enter the cell, reverse transcribe the viral RNA into DNA, enter the nucleus, and integrate into host cell chromatin. Studies in Project 1, Infecting the Cell, will focus on defining these key steps in the first half of the HIV-1 life cycle in molecular and mechanistic detail. A particular theme will be to elucidate the organization, functional roles, and dynamic transformations of the viral core particle. Studies in Aim 1 (Virus Entry) will build on our previous structural studies of different states of HIV-1 Env and imaging of viral entry complexes in situ to provide structural details of the virus-target cell interaction at the time of entry including: 1) the number of Envs and average spacing in a typical interaction with a target cell, 2) whether there is rearrangement of the Env subunits that are not bound to host receptors, and 3) the structure of the hypothesized pre-hairpin intermediate formed between the virus and host cell membranes by Env and host receptor(s) at sufficient resolution to dock coordinates from components of known structure. Studies in Aim 2 (Genome Structure and Reverse Transcription Initiation) will build on our previous structures of HIV-1 replication initiation complexes to reveal: 1) the structural basis for the transition from initiation to elongation, 2) how the dimeric structure of the viral RNA modulates reverse transcription initiation, 3) how the global dynamic architecture of HIV-1 genomic RNA is influenced by the capsid, and 4) how this dynamic architecture influences the process of reverse transcription, including strand jumps. Studies in Aim 3 (Capsid Functions in Infection) will build on our development of a cell-free system that efficiently reconstitutes the coupled processes of viral endogenous reverse transcription and integration to: 1) characterize the viral genomic template at different stages of reverse transcription, 2) reveal how the capsid facilitates reverse transcription, 3) define how the potent new capsid inhibitor, lenacapavir, binds the capsid and inhibits nuclear entry, and 4) reconstitute and image core passage through nuclear pores. Studies in Aim 4 (Core Uncoating and Integration) will build on our cell-free integration assay and our ability to reconstruct viral capsid lattices at high resolution to fill a series of fundamental knowledge gaps, including: 1) the identities and functional roles of host factors that promote uncoating and integration, 2) how the viral capsid “opens” to expose the integrating viral DNA, and 3) the 3D architectures of active, native intasomes.

项目摘要 为了引发感染，HIV-1必须进入细胞，将病毒RNA逆转录为DNA，进入细胞核， 整合到宿主细胞染色质中。项目1的研究，感染细胞，将集中在定义这些关键步骤，在第一个 HIV-1生命周期的一半分子和机制细节。一个特别的主题将是阐明该组织， 功能作用和病毒核心颗粒的动态转化。 目标1（病毒进入）的研究将建立在我们以前对HIV-1 Env不同状态的结构研究和对 原位病毒进入复合物，以提供进入时病毒-靶细胞相互作用的结构细节，包括： 1)在与靶细胞的典型相互作用中Env的数量和平均间距，2）是否存在 不与宿主受体结合的Env亚基，和3）假设的前发夹中间体的结构 在病毒和宿主细胞膜之间由Env和宿主受体以足够的分辨率形成，以对接坐标 来自已知结构的组件。 目标2（基因组结构和逆转录起始）的研究将建立在我们以前的HIV-1结构的基础上 复制起始复合物，以揭示：1）从起始过渡到延伸的结构基础，2） 病毒RNA的二聚体结构调节逆转录起始，3）病毒RNA的全局动态结构如何调节逆转录起始， HIV-1基因组RNA受衣壳的影响，以及4）这种动态结构如何影响逆转过程 转录，包括链跳跃。 目标3（感染中的衣壳功能）的研究将建立在我们开发的无细胞系统的基础上， 重组了病毒内源性逆转录和整合的偶联过程，以：1）表征病毒 逆转录不同阶段的基因组模板，2）揭示衣壳如何促进逆转录，3） 定义有效的新衣壳抑制剂lenacapavir如何结合衣壳并抑制核进入，以及4）重建和 通过核孔成像核心通道。 目标4（核心去包衣和整合）的研究将建立在我们的无细胞整合试验和我们重建 高分辨率的病毒衣壳晶格，以填补一系列基本知识空白，包括：1）身份和 促进脱壳和整合的宿主因子的功能作用，2）病毒衣壳如何“打开”以暴露病毒衣壳， 整合病毒DNA，和3）活性的天然整合体的3D结构。