Spatiotemporal Regulation of Membrane Raft Trafficking in Virus Activated Cells

描述（由申请方提供）：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）。缺乏预防或治疗HCPS疾病的疫苗或特效药，以及需要在生物安全三级实验室（BSL-3）进行实验，限制了有效探索感染机制和疾病发病机制的能力。因此，我们已经启动了一项计划，以研究用校准剂量的UV辐射杀死的Sin Nombre病毒（SNV）作为模型系统，以剖析其在BSL-2设施中进入细胞的机制。作为专性细胞内寄生虫，汉坦病毒依赖于细胞机制进入和从其宿主细胞释放。细胞表面的膜筏通常是细胞信号传导、粘附和内吞的起点。病毒使用捆绑在膜筏中的信号元件，并协同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，并且没有可利用的反向遗传学系统，因此病毒进入的机械解剖受到阻碍。因此，需要新的方法来理解感染的机制步骤，并且对于确定阻断感染的靶点至关重要。我们证明，紫外线杀死的SNV附着在糖基磷脂酰肌醇（GPI）锚定的蛋白衰变加速因子（CD 55）和低亲和力状态？3整联蛋白，其方式与使用活病毒体的感染性测定获得的结果平行。因此，我们能够监测病毒摄取在真实的时间，并表明，SNV的初始结合到Rac 1刺激肌动蛋白重塑，解体的焦点粘附，并导致细胞-细胞接触和细胞-基质粘附的损失。我们的观察是有意义的，因为他们提供了第一个时间和空间解剖的汉他病毒进入所涉及的事件，并提供了一个合理的机制解释的急性心肺综合征引起的病毒感染。为了检验这个假设，我们将追求以下目标。目的1：确定同源受体（CD 14/CD 55和？3整联蛋白）。目的2：阐明汉坦病毒内吞作用的机制及其与细胞增殖的关系。3、。 公共卫生相关性：汉坦病毒心肺综合征是一种进行性的，往往是致命的人类疾病，由辛诺布尔病毒（SNV）引起，这是一种啮齿动物病毒，流行于美国西南部。这项建议汇集了一个独特的团队，采用生物物理科学，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研究病毒如何劫持细胞内吞机制，通过称为脂筏的专门膜微区的介导进入和感染细胞。这项工作的长期结果可用于确定抗病毒治疗的进入途径。

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.