Biophysics of Protein-Mediated Membrane Fusion

蛋白质介导的膜融合的生物物理学

• 批准号: 9538192
• 负责人: Gregory B Melikian
• 金额: $ 46.92万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9538192
• 关键词: Actins; Biological Assay; Biophysics; CCR5 gene; CD4 Positive T Lymphocytes; CXCR4 gene; Cell fusion; Cell membrane; Cell surface; Cells; Cytoplasm; DNA; Dependence; Dynamin; Electron Microscopy; Endosomes; Exhibits; Fluorescence; Foundations; Future; Goals; Growth; HIV; HIV-1; Image; Immobilization; Infection; Infection prevention; Integration Host Factors; Knowledge; Label; Laboratories; Light; Location; Mechanics; Mediating; Membrane; Membrane Fusion; Microscopy; Modeling; Molecular Conformation; Monitor; Process; Property; Proteins; Regulation; Resolution; Role; Signal Transduction; Site; Solid; Stress; Therapeutic; Viral; Virus; biophysical tools; blocking factor; env Glycoproteins; experimental study; mechanical force; novel; novel strategies; particle; prevent; protein transport; small hairpin RNA; spatiotemporal

HIV-1 infection is initiated through fusion with a cell membrane, a process that is mediated by Env glycoprotein following the engagement of CD4 and coreceptors. However, there is increasing appreciation that the HIV-1 fusion process is regulated by host dependency and restriction factors, including the newly discovered SERINC5 protein that potently blocks virus entry. This realization suggests that the availability and/or abundance of host regulatory factors could determine the efficiency of HIV-1 fusion, as well as the sites of productive entry. Indeed, whereas HIV-1 fusion initiated at the cell surface does not appear to proceed to completion, this virus can mediate cell-cell fusion (“fusion-from-without”) by fusing with the plasma membranes of two adjacent cells. Our recent study has demonstrated that fusion-from-without, but not HIV-cell fusion, is highly dependent on actin dynamics, suggesting that fusion with the plasma membrane is driven by cell- generated mechanical forces. We therefore hypothesize that HIV-1 Env initiates membrane fusion but relies on a target cell to drive the energetically unfavorable enlargement of a fusion pore. This model suggests a universal mechanism for inhibition of HIV-1 fusion by SERINC5 and other restriction factors that could act through altering the properties of virus or cell membranes. The knowledge of underlying principles for promotion or inhibition of HIV-1 entry is of great importance for preventing infection and for future therapeutic strategies. Our goal is to explore the vulnerabilities of HIV-1 fusion and exploit those to develop new means to block virus entry. We propose to develop and apply novel biophysical tools to detect fusion pore dilation, delineate the determinants of productive fusion and reveal the key host factors and processes involved. Specifically, we will: (1) Develop cryo-CLEM strategies to assess the effect of HIV-1 entry sites on dilation of a fusion pore; (2) Elucidate the role of cell-generated mechanical forces in completing HIV-1 fusion at the plasma membrane and in endosomes; and (3) Delineate the mechanism by which SERINC5 inhibits HIV-1 fusion. Completion of the proposed experiments will help define the mechanisms of regulation of HIV-1 fusion (and viral fusion in general) and pave the way to developing novel strategies to prevent and treat infection.

HIV-1感染是通过与细胞膜融合而开始的，这一过程由Env糖蛋白介导 在CD 4和辅助受体的接合之后。然而，越来越多的人认识到， 融合过程受宿主依赖性和限制性因素的调节，包括新发现的 SERINC 5蛋白，有效阻止病毒进入。这一认识表明，可用性和/或 宿主调节因子的丰度可以决定HIV-1融合的效率，以及融合的位点。 生产性进入。事实上，尽管HIV-1融合在细胞表面开始，但似乎不会继续进行， 完成后，该病毒可以通过与质膜融合来介导细胞-细胞融合（“从无融合”）。 两个相邻的细胞。我们最近的研究表明，从没有融合，但不是艾滋病毒细胞融合， 高度依赖于肌动蛋白动力学，表明与质膜的融合是由细胞驱动的， 产生机械力。因此，我们假设HIV-1 Env启动膜融合，但依赖于 靶细胞来驱动融合孔的能量上不利的扩大。该模型表明， SERINC 5和其他限制性因子抑制HIV-1融合的普遍机制 通过改变病毒或细胞膜的特性。基本原则的知识 促进或抑制HIV-1进入对于预防感染和未来的治疗非常重要。 战略布局我们的目标是探索HIV-1融合的弱点，并利用这些弱点开发新的方法， 阻止病毒进入。我们建议开发和应用新的生物物理工具来检测融合孔扩张， 描述了生产融合的决定因素，并揭示了关键的主机因素和过程。 具体来说，我们将：（1）发展cryo-CLEM策略，以评估HIV-1进入位点对扩张的影响。 （2）阐明细胞产生的机械力在完成HIV-1在血浆中融合中的作用 （3）阐明SERINC 5抑制HIV-1融合的机制。 完成拟议的实验将有助于确定HIV-1融合的调节机制（和 病毒融合一般），并为开发预防和治疗感染的新策略铺平道路。