Spatiotemporal Regulation of Membrane Raft Trafficking in Virus Activated Cells

病毒激活细胞膜筏运输的时空调控

• 批准号: 8028735
• 负责人: TIONE BURANDA
• 金额: $ 7.54万
• 依托单位: UNIVERSITY OF NEW MEXICO HEALTH SCIS CTR
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-12-15 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8028735
• 关键词: Actins; Acute; Adhesions; Affinity; American; Beryllium; Binding; Biochemical; Biological Assay; Biological Models; CD55 Antigens; Cardiopulmonary; Cell surface; Cells; Cellular biology; Clathrin; Collaborations; Development; Disease; Dissection; Dose; Endocytosis; Event; FDA approved; Fluorescent Probes; Focal Adhesions; GPI Membrane Anchors; Glycoproteins; Hantavirus; Hemorrhagic Fever with Renal Syndrome; Infection; Integrins; Label; Laboratories; Lead; Life; Link; Lipids; Measurement; Mediating; Mediation; Membrane; Membrane Microdomains; Methods; Microscopy; Molecular; Monitor; Movement; Organelles; Pathogenesis; Pathway interactions; Pattern; Pharmaceutical Preparations; Positioning Attribute; Proteins; Reagent; Recruitment Activity; Regulation; Research; Rodent; Route; Science; Signal Transduction; Sin Nombre virus; Site; Syndrome; System; Technology; Testing; Time; Titrations; Translating; Ultraviolet Rays; Vaccines; Viral; Virion; Virus; Virus Diseases; Virus Receptors; Work; base; biosafety level 2 facility; crosslink; experience; high throughput screening; human disease; inhibitor/antagonist; innovation; killings; novel strategies; obligate intracellular parasite; particle; polarized cell; positional cloning; prevent; programs; receptor; research study; small molecule; spatiotemporal; tool; trafficking; uptake; viral RNA; virology

DESCRIPTION (provided by applicant): Hantaviruses can cause two often-severe diseases of humans: hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome (HCPS). The lack of vaccines or specific drugs to prevent or treat HCPS disease, and the requirement for conducting experiments in a biosafety level 3 laboratory (BSL-3), have limited the ability to effectively probe the mechanism of infection and disease pathogenesis. We have therefore initiated a program to study Sin Nombre virus (SNV) killed with a calibrated dose of UV radiation as a model system to dissect its mechanism of cellular entry in BSL-2 facilities. As obligate intracellular parasites, hantaviruses depend on cellular mechanisms for entry and release from their host cells. Membrane rafts on the cell surface are often the originating point of cell signaling, adhesion and endocytosis. Viruses use signaling elements that are bundled in membrane rafts and co-opt the cellular endocytic machinery to achieve entry and productive infection. However, mechanistic dissection of virus entry has been hampered because glycoproteins of many hantaviruses are difficult to express and no tractable reverse genetics system is available. Therefore, new approaches to understanding the mechanistic steps of infection are needed and critical to the identification of targets for blocking infection. We demonstrated that UV-killed SNV attaches to the glycosylphosphatidylinositol (GPI)-anchored protein decay accelerating factor (DAF/CD55) and low affinity state ???3 integrins in a manner that parallels the results obtained from infectivity assays using live virions. Therefore, we were able to monitor virus uptake in real time and show that the initial binding of SNV to DAF is followed by Rac1 stimulated actin remodeling, disassembly of focal adhesions, and leads to loss of cell-cell contact and cell-substrate adhesion. Our observations are significant because they offer the first temporal and spatial dissection of the events involved in hantavirus entry and offer a plausible mechanistic explanation for the acute cardiopulmonary syndrome caused by virus infection. To test this hypothesis we will pursue the following aims. Aim 1: To define the spatiotemporal redistribution of cognate receptors (DAF/CD55 and ???3 integrins) following hantavirus binding. Aim 2: To define the mechanism of hantavirus endocytosis and its relationship to ???3 and DAF. PUBLIC HEALTH RELEVANCE: Hantavirus cardiopulmonary syndrome is a progressive and often fatal form of human disease caused by Sin Nombre Virus (SNV), which is a rodent virus endemic to the American Southwest. This proposal brings together a unique team employing tools and concepts of biophysical science, molecular cell biology and virology to study the mechanism used by hantaviruses to enter cells. We are able to use state of the art tools to examine how viruses hijack the cellular machinery of endocytosis to enter and infect cells through the mediation of specialized membrane microdomains called lipid rafts. The long-term result of this work could be used to determine route of entry for anti-viral treatment.

描述(申请人提供)：汉坦病毒可导致人类两种经常严重的疾病：肾综合征出血热或汉坦病毒心肺综合征(HCPS)。缺乏预防或治疗HCPS疾病的疫苗或特定药物，以及需要在生物安全3级实验室(BSL-3)进行实验，限制了有效探索感染机制和疾病发病机制的能力。因此，我们启动了一个项目，研究校准剂量的紫外线辐射杀死的新诺布雷病毒(SNV)，作为一个模型系统，以剖析其进入BSL-2设施的细胞机制。作为专性的细胞内寄生虫，汉坦病毒依靠细胞机制进入和释放它们的宿主细胞。细胞表面的膜筏通常是细胞信号、黏附和内吞作用的起始点。病毒使用捆绑在膜筏中的信号元件，并利用细胞内吞机制来实现进入和生产性感染。然而，由于许多汉坦病毒的糖蛋白很难表达，而且没有易于处理的反向遗传学系统，因此对病毒进入的机制解剖一直受到阻碍。因此，需要新的方法来了解感染的机制步骤，这对于确定阻断感染的靶点至关重要。我们证明了紫外线灭活的SNV与糖基磷脂酰肌醇(GPI)锚定的蛋白衰变加速因子(DAF/CD55)和低亲和力状态的整合素结合的方式与使用活病毒粒子进行感染性检测的结果相似。因此，我们能够实时监测病毒摄取，并表明SNV与DAF的初始结合之后是rac1刺激的肌动蛋白重塑，局部黏附的解体，并导致细胞-细胞接触和细胞-底物黏附的丧失。我们的观察具有重要意义，因为它们首次对汉坦病毒进入过程中涉及的事件进行了时间和空间上的剖析，并为病毒感染引起的急性心肺综合征提供了可信的机制解释。为了验证这一假设，我们将追求以下目标。目的1：明确汉坦病毒结合后同源受体(DAF/CD55和β3整合素)的时空再分布。目的2：明确汉坦病毒内吞作用的机制及其与β-3和DAF的关系。 公共卫生相关性：汉坦病毒心肺综合征是由新诺布雷病毒(SNV)引起的一种进行性且往往是致命的人类疾病，SNV是美国西南部的一种啮齿动物病毒。这项建议汇集了一个独特的团队，利用生物物理学、分子细胞生物学和病毒学的工具和概念来研究汉坦病毒进入细胞的机制。我们能够使用最先进的工具来检查病毒如何劫持内吞作用的细胞机制，通过称为脂筏的特殊膜微域的中介进入并感染细胞。这项工作的长期结果可用于确定抗病毒治疗的进入途径。