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）：汉坦病毒可引起两种常见的严重人类疾病：肾综合征出血热或汉坦病毒心肺综合征（HCPS）。由于缺乏预防或治疗HCPS疾病的疫苗或特效药物，以及需要在生物安全三级实验室（BSL-3）进行实验，限制了有效探讨感染机制和疾病发病机制的能力。因此，我们启动了一项计划，研究用校准剂量的紫外线辐射杀死的 Sin Nombre 病毒 (SNV)，作为模型系统来剖析其进入 BSL-2 设施的细胞机制。作为专性细胞内寄生虫，汉坦病毒依赖于细胞机制进入宿主细胞和从宿主细胞释放。细胞表面的膜筏通常是细胞信号传导、粘附和内吞作用的起点。病毒利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难以表达并且没有可用的反向遗传学系统，病毒进入的机械解剖受到阻碍。因此，需要新的方法来了解感染的机制步骤，这对于识别阻止感染的目标至关重要。我们证明，紫外线灭活的 SNV 附着在糖基磷脂酰肌醇 (GPI) 锚定的蛋白质衰变加速因子 (DAF/CD55) 和低亲和力态 ???3 整联蛋白上，其方式与使用活病毒粒子的感染性测定获得的结果相似。因此，我们能够实时监测病毒摄取，并表明 SNV 与 DAF 的初始结合随后是 Rac1 刺激的肌动蛋白重塑、粘着斑分解，并导致细胞与细胞接触和细胞与基质粘附的丧失。我们的观察很重要，因为它们首次对汉坦病毒进入所涉及的事件进行了时间和空间剖析，并为病毒感染引起的急性心肺综合征提供了合理的机制解释。为了检验这一假设，我们将追求以下目标。目标 1：确定汉坦病毒结合后同源受体（DAF/CD55 和 ???3 整合素）的时空重新分布。目标 2：明确汉坦病毒内吞作用的机制及其与 ???3 和 DAF 的关系。 公共卫生相关性：汉坦病毒心肺综合征是一种进行性且常常致命的人类疾病，由辛诺布尔病毒 (SNV) 引起，辛诺布尔病毒是美国西南部流行的一种啮齿动物病毒。该提案汇集了一个独特的团队，利用生物物理科学、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细胞内吞作用机制，通过称为脂筏的特殊膜微域的介导进入并感染细胞。这项工作的长期结果可用于确定抗病毒治疗的进入途径。