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The Role of the Actin Cytoskeleton and Cholesterol in Influenza Virus Assembly

肌动蛋白细胞骨架和胆固醇在流感病毒组装中的作用

基本信息

批准号：
7980666
负责人：
SAMUEL T HESS
金额：
$ 42.49万
依托单位：
UNIVERSITY OF MAINE ORONO
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-01 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7980666
关键词：
Actins Adaptor Signaling Protein Affect Aftercare Annexins Binding Biological Assay Cell Membrane Structures Cell membrane Cells Cholesterol Cluster Analysis Communicable Diseases Cultured Cells Cytoskeleton Data Development Diffusion Drug Delivery Systems Electron Microscopy Fluorescence Fluorescence Microscopy Fluorescence Resonance Energy Transfer Health Hemagglutinin Human Image Imaging Techniques Individual Infection Influenza Influenza A Virus, H1N1 Subtype Investigation Length Life Light Mass Spectrum Analysis Mediating Membrane Membrane Microdomains Membrane Proteins Microscopy Morphology Neuraminidase Pharmaceutical Preparations Physiological Proteins Resistance Resolution Role Structure Techniques Temperature Testing Time Tubulin Variant Viral Viral Matrix Proteins Viral Proteins Virus Virus Assembly Virus Diseases cellular imaging influenzavirus inhibitor/antagonist membrane model millisecond nanometer novel photoactivation prophylactic protein distribution public health relevance response single molecule

项目摘要

DESCRIPTION (provided by applicant): Influenza drugs are few in number, and novel strains of the virus are resistant to many existing drugs. Thus, new drug targets are desperately needed. Influenza virus depends on cell membrane organization for entry through hemagglutinin-mediated fusion, and for assembly of viral components such as hemagglutinin (HA) and neuraminidase (NA) before budding. Cell membrane organization depends on cholesterol-rich membrane domains called "rafts," with which HA is associated, but the structure of these domains has been difficult to image directly because of the limits of light and electron microscopy for live cell imaging at nanometer resolution. Preliminary results show that HA clustering depends both on cholesterol and cortical actin, both of which are known to mediate cell membrane organization. We hypothesize that HA and NA clustering depend on cholesterol and cortical actin, and we will test this hypothesis using a novel super- resolution microscopy technique called fluorescence photoactivation localization microscopy (FPALM), developed in the Hess lab in 2006. FPALM will allow us to image individual molecules of HA or NA and actin cytoskeletal components, including actin- binding adaptor proteins, in living cells at physiological temperature, with resolution in the 10-40 nanometer range. Along with actin and tubulin, the candidate adaptor proteins were found in purified influenza virus as observed by mass spectroscopy. We will also test the hypothesis that HA and NA membrane clustering is mediated by interactions with one or more of these adaptor proteins. Finally, since the membrane organization of "rafts" is dependent on cholesterol and the cytoskeleton, we will test the hypothesis that drugs which reduce cell cholesterol and disrupt the actin cytoskeleton will affect HA and NA membrane clustering. The observation of a significant response to such drugs would constitute a major opportunity for development of a new class of drugs against influenza. PUBLIC HEALTH RELEVANCE: Cell membrane cholesterol and the cytoskeleton affect influenza membrane protein clustering needed for infection, so drugs which alter cholesterol and the cytoskeleton are good candidates as anti-viral therapies. We will use super-resolution fluorescence microscopy to test for interactions between viral components and the cytoskeleton, and to test the effects of these drugs on the assembly of viral components into membrane clusters. Drugs identified to have effects on viral protein clustering could be used as anti- viral therapies to reduce the spread of new strains of influenza and other viruses.

描述（由申请人提供）：流感药物的数量很少，新的病毒株对许多现有药物具有耐药性。因此，迫切需要新的药物靶点。流感病毒依赖于细胞膜组织通过血凝素介导的融合进入，并在出芽前组装病毒组分，如血凝素（HA）和神经氨酸酶（NA）。细胞膜组织依赖于称为“筏”的富含胆固醇的膜结构域，HA与之相关，但这些结构域的结构难以直接成像，因为光和电子显微镜在纳米分辨率下用于活细胞成像的限制。初步结果表明，HA聚类依赖于胆固醇和皮质肌动蛋白，这两者都是已知的介导细胞膜组织。我们假设HA和NA聚集依赖于胆固醇和皮质肌动蛋白，我们将使用2006年在Hess实验室开发的称为荧光光活化定位显微镜（FPAL）的新型超分辨率显微镜技术来验证这一假设。FPALM将使我们能够在生理温度下对活细胞中的HA或NA和肌动蛋白细胞骨架组分（包括肌动蛋白结合接头蛋白）的单个分子进行成像，分辨率在10-40纳米范围内。通过质谱观察，在纯化的流感病毒中发现了沿着肌动蛋白和微管蛋白的候选衔接蛋白。我们还将测试的假设，HA和NA膜簇介导的相互作用与这些衔接蛋白。最后，由于膜组织的“筏”是依赖于胆固醇和细胞骨架，我们将测试的假设，即药物降低细胞胆固醇和破坏肌动蛋白细胞骨架将影响HA和NA膜集群。观察到对这些药物的显著反应将为开发一类新的抗流感药物提供重大机会。公共卫生相关性：细胞膜胆固醇和细胞骨架影响感染所需的流感膜蛋白聚集，因此改变胆固醇和细胞骨架的药物是抗病毒治疗的良好候选者。我们将使用超分辨率荧光显微镜来测试病毒成分和细胞骨架之间的相互作用，并测试这些药物对病毒成分组装成膜簇的影响。经鉴定对病毒蛋白质聚集有影响的药物可用作抗病毒疗法，以减少流感和其他病毒新菌株的传播。