Determining the role of microtubules and motor proteins during early HIV-1 replication

确定微管和运动蛋白在早期 HIV-1 复制过程中的作用

• 批准号: 10161131
• 负责人: Michael Cianfrocco
• 金额: $ 24.24万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-18 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10161131
• 关键词: Acquired Immunodeficiency Syndrome; Address; Affect; Behavior; Binding; Biological Assay; Biology; Capsid; Cell Nucleus; Cell membrane; Cells; Complex; Crowding; Cytoplasm; Cytoskeleton; DNA; Data; Diffusion; Docking; Dominant-Negative Mutation; Dynein ATPase; Engineering; Equilibrium; Exhibits; Fluorescence; Fluorescence Microscopy; Foundations; Future; Genome; Goals; HIV-1; In Vitro; Individual; Infection; Infection prevention; Integration Host Factors; Kinesin; Label; Measures; Mediating; Membrane; Membrane Fusion; Microtubules; Monitor; Motor; Motor Activity; Movement; Nuclear; Nuclear Translocation; Process; Proteins; Reporting; Research; Role; Structure; System; Therapeutic; Viral; Virus; Walking; Work; base; cell motility; live cell imaging; novel; reconstitution; recruit; scaffold; single molecule; trafficking

Determining the role of microtubules and motor proteins during early HIV-1 replication SUMMARY During HIV-1 infection, viral membrane fuses with the host cell membrane to deliver the HIV-1 virus to the host cell cytoplasm. Once within the cell, HIV-1 must translocate to the nucleus for the reverse transcribed DNA to be incorporated into the host genome. Despite 40+ years of research devoted to HIV-1 infection, surprisingly little is known about how HIV-1 exploits the host cell cytoskeleton to facilitate transport to the nucleus and accelerate infection. The goal of this proposal is to establish and validate an assay to measure HIV-1 microtubule trafficking in vitro via kinesin and dynein microtubule motor proteins. Recent work has led to the identification of two host factors - FEZ1 and BicD2 - that serve as cargo adaptors for transport via kinesin-1 and cytoplasmic dynein-1, respectively. To study the role of FEZ1 and BicD2 in the transport of HIV-1, we will utilize single-molecule total internal reflection fluorescence (TIRF) microscopy to monitor the transport of fluorescently-labeled virus-motor complexes on reconstituted microtubules (Aim 1). We will use a novel synthetic icosahedral scaffold derived from encapsulins to validate the assay, which we will use to measure the motility of motor protein teams bound to viral cargo via cargo adaptors. We will then determine how HIV-1 determines directionality on microtubules (kinesin vs. dynein) by measuring the combined influences of FEZ1, BicD2, kinesin-1, and dynein (Aim 2). By establishing that microtubule motors are capable of transporting HIV-1 in vitro, this proposal will provide a new avenue in the study of HIV-1 and provide new targets to block HIV-1 infection.

确定微管和马达蛋白在HIV-1早期复制中的作用 总结 在HIV-1感染过程中，病毒膜与宿主细胞膜融合，将HIV-1病毒递送给宿主 细胞质一旦进入细胞，HIV-1必须转移到细胞核，以便逆转录DNA， 被整合到宿主基因组中。尽管40多年来致力于HIV-1感染的研究，令人惊讶的是， 关于HIV-1如何利用宿主细胞的细胞骨架促进转运到细胞核， 加速感染。该提案的目标是建立和验证一种检测HIV-1的方法 通过驱动蛋白和动力蛋白微管马达蛋白的体外微管运输。最近的工作导致了 两种宿主因子FEZ 1和BicD 2作为通过驱动蛋白1转运的货物衔接子的鉴定 和细胞质动力蛋白-1。为了研究FEZ 1和BicD 2在HIV-1转运中的作用，我们将 利用单分子全内反射荧光（TIRF）显微镜监测运输 荧光标记的病毒-马达复合物在重构的微管上（Aim 1）。我们会用一本小说 合成的二十面体支架衍生自白蛋白，以验证该测定，我们将使用该测定来测量 通过货物衔接子与病毒货物结合的马达蛋白组的运动性。然后我们将确定HIV-1 通过测量FEZ 1的组合影响来确定微管上的方向性（驱动蛋白与动力蛋白）， BicD 2、驱动蛋白-1和动力蛋白（Aim 2）。通过确定微管马达能够运输 HIV-1的体外研究，这一建议将为HIV-1的研究提供一条新的途径，并提供新的靶点来阻断 HIV-1感染。