Spatiotemporal Regulation of Membrane Raft Trafficking in Virus Activated Cells

点击翻译按钮获取中文摘要

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.