Elucidating Non-Virus Encoded HIV Capture through Artificial Virus Nanoparticles

描述（由申请人提供）：最近的研究表明，GM 3（一种含有末端连接的唾液酸残基的神经节苷脂）可以介导人免疫缺陷病毒1（HIV-1）和树突状细胞（DC）之间的糖蛋白非依赖性相互作用，因此可以诱导DC对病毒的包膜糖蛋白非依赖性捕获。这个重要的捕获机制的许多方面仍然未知，其系统的调查是缺乏适当的工具来表征病毒膜中的GM 3的浓度和空间分布和具有明确定义的表面组成的适当的病毒模型系统的挑战。该提案旨在通过开发新的基于贵金属纳米颗粒的工具来克服这些挑战，以研究非病毒编码的表面功能在HIV-1发病机制中的作用。我们将利用贵金属纳米颗粒独特的电磁特性来量化GM 3的表面密度及其在来自外周血单核细胞（PBMC）的病毒上的空间分布。关于HIV-1颗粒中GM 3含量的定量信息将形成实施人工病毒纳米颗粒（AVN）的基础，所述人工病毒纳米颗粒包含包裹在磷脂双层膜中的金核心，所述磷脂双层膜的脂质组成模拟天然病毒的脂质组成。作为人造的、可工程化的纳米颗粒，可以调节AVN GM 3表面 在一些实施方案中，本发明的组合物可以以系统的方式浓缩，而没有包括任何其他宿主或病毒编码的表面官能团的风险。本文介绍的AVN将金属NP的有利材料性质与生物膜的可编程功能性联合收割机结合，方便地大量产生，在荧光和暗场光学显微镜中提供大的光学横截面，并且可以在电子和光学显微镜中以高分辨率定位。因此，AVN是确定GM 3在DC上捕获HIV-1颗粒中的贡献的理想工具，并且将使我们能够探测GM 3依赖的HIV-1入侵DC的复杂机制。本申请的具体目的是：目的1：使用纳米颗粒使能的测定来确定GM 3在原代HIV-1分离物与DC的相互作用中的作用目的2：开发模拟GM 3介导的HIV-1捕获的人工病毒纳米颗粒（AVN）目的3：使用AVN阐明GM 3-CD 169介导的DC内HIV-1颗粒的结合和摄取的分子机制。

DESCRIPTION (provided by applicant): Recent studies have indicated that GM3 (a ganglioside that contains a terminal ¿-2,3 linked sialic acid residue) can mediate glycoprotein independent interactions between human immunodeficiency virus 1 (HIV-1) and dendritic cells (DCs) and can, thus, induce an envelope glycoprotein-independent capture of the virus by DCs. Many aspects of this important capture mechanism remain unknown and its systematic investigation is challenged by a lack of appropriate tools to characterize the concentration and spatial distribution of GM3 in the virus membrane and of appropriate virus model systems with clearly defined surface composition. This proposal seeks to overcome these challenges by developing new noble metal nanoparticle-based tools for investigating the role of non-virus encoded surface functionalities in the pathogenesis of HIV-1. We will take advantage of the unique electromagnetic properties of noble metal nanoparticles to quantify the - as yet unknown - surface density of GM3 and its spatial distribution on viruses derived from peripheral blood mononuclear cells (PBMCs). Quantitative information about the GM3 content in HIV-1 particles will form the basis for the implementation of artificial virus nanoparticles (AVNs) that contain a gold core wrapped in a phospholipid bilayer membrane whose lipid composition mimics that of the native virus. As artificial, engineerable nanoparticles, it is possible to tune AVN GM3 surface concentration in a systematic fashion without the risk of including any other host or virus encoded surface functionalities. The AVNs introduced herein combine the advantageous material properties of metal NPs with the programmable functionality of biological membranes, are conveniently generated in large quantities, provide large optical cross-sections in fluorescent and darkfield optical microscopy, and can be localized with high resolution in both electron and optical microscopy. AVNs are, thus, ideal tools to determine the contribution of GM3 in the capture of HIV-1 particles on DCs and will enable us to probe the probe the intricate mechanisms underlying the GM3- dependent HIV-1 invasion of DCs. The specific aims of this application are: Aim1: To define the role of GM3 in interactions of primary HIV-1 isolates with DCs using nanoparticle enabled assays Aim2: To develop artificial virus nanoparticles (AVNs) that mimic GM3-mediated HIV-1 capture Aim3: To elucidate the molecular mechanisms underlying GM3-CD169 mediated binding and uptake of HIV-1 particles within DCs using AVNs.