Nanoparticle Analysis of Enveloped Virus Entry Pathways

描述（由申请人提供）：在纳米技术领域，构建可容纳药物和其他治疗剂的纳米级颗粒受到了广泛关注。然而，除了使用静态配体（如抗体和小肽）之外，对纳米颗粒（NPs）的特异性靶向研究很少。这是NP设计的重要组成部分。通过选择性地将药物传递到一个细胞而不是另一个细胞，药物的功效可以大大提高，从而减少所需的剂量和潜在的副作用。病毒在结构和物理上与理想NP有许多相似之处。它们的直径都在100纳米左右，具有亲水性外壳，循环寿命长。然而，病毒实现了很少有NP设计能做到的壮举；它们利用病毒包膜蛋白（envs）有效地靶向特定组织和细胞。嫉妒体还做了一些设计配体可能永远做不到的事情；它们以特定的细胞内吞途径为目标，进入高尔基体和内质网等细胞器。Envs也是一种简单的膜融合引擎，它能感知周围的环境，从而在细胞质的特定位置触发其货物的释放。在本课题中，我们将优化在Friend小鼠白血病病毒、委内瑞拉马脑炎病毒（NIH B类病原体）和埃博拉病毒（NIH a类病原体）的眼膜中构建荧光NPs的技术。每个环境都将病毒靶向到独特的细胞受体和内吞进入途径。我们将使用最先进的共聚焦显微镜和生化方法研究包被的envs的细胞和细胞器靶向特性。结合一种靶向NPs的新方法，这项工作将使人们更好地了解envs在感染中的作用以及每种病毒使用的进入途径。这将确定抗病毒药物开发的新靶点。NPs在快速病毒诊断分析中也很有用。

DESCRIPTION (provided by applicant): In the field of nanotechnology, much attention has been paid to the construction of nanometer- sized particles that can harbor drugs and other therapeutic agents. However, beyond the use of static ligands such as antibodies and small peptides, little work has been done on the specific targeting of the nanoparticles (NPs). This is an important component of NP design. By selectively delivering drugs to one cell over another, the efficacy of the drug can be drastically improved and consequently reduce the dosage needed and potential side effects. Viruses share many structural and physical similarities to an idealized NP. They are both about 100 nm in diameter, have hydrophilic shells and have long circulatory lives. However, viruses achieve a feat that few NP designs can; they efficiently target specific tissues and cells using virus envelope proteins (envs). The envs also do something that designed ligands may never achieve; they target specific cellular endocytic pathways, to access organelles such as the Golgi and the endoplasmic reticulum. Envs are also simple membrane fusion engines that sense the surrounding environment to trigger the release of their cargo in the cell cytoplasm at specific sites. In this proposal, we will optimize techniques to construct fluorescent NPs coated in the envs of Friend murine leukemia virus, Venezuelan equine encephalitis virus (NIH category B agent) and Ebola (NIH category An agent). Each env targets virus to unique cell receptors and endocytic entry pathways. We will investigate the cell and organelle targeting properties of the coated envs using state-of-the-art confocal microscopy and biochemical methods. Together with a novel way to target NPs, this work will allow a better understanding of the role of envs in infection and the entry pathways used by each virus. This will identify new targets for antiviral drug development. The NPs will also be useful in rapid virus diagnostic assays. Project Narrative: Highly purified virus membranes will be used to coat nanoparticles using a new method that preserves the useful features of the virus envelope proteins. The nanoparticles will then be tested for their ability to specifically interact with and enter cells. This will greatly aid in the development of targeted drug therapies and vaccine design.