"Investigation of Membrane Fusion Interactions of Enveloped Viruses using Magnetically-Labeled Liposomes"

“使用磁性标记脂质体研究包膜病毒的膜融合相互作用”

基本信息

批准号：
10439023
负责人：
Santimukul Santra
金额：
$ 7.69万
依托单位：
PITTSBURG STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-01 至 2023-10-23
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10439023
关键词：
2019-nCoV ACE2 Affinity Antiviral Agents Biological Biological Assay Biomedical Research Cell Line Cell fusion Cell membrane Cells Characteristics Chimeric Proteins Coupling Data Data Analyses Development Disease Outbreaks Dyes Ebola Environmental Risk Factor Evaluation Fluorescence Ganglioside GM1 Gangliosides Glycoproteins Goals Hemagglutinin Image Analysis Infection Influenza Institution Investigation Label Learning Lipids Liposomes Luciferases Magnetism Mammalian Cell Measurement Measures Mediating Membrane Membrane Fusion Membrane Lipids Methods Modeling Molecular Conformation Outcomes Research Pathogen detection Pathogenicity Peptide Hydrolases Phase Play Principal Investigator Process Property Proteins Rapid screening Reaction Recombinants Relaxation Relaxation Techniques Reporter Reporting Reproducibility Role Screening procedure Sialic Acids Signal Transduction Students Technology Temperature Testing Time Trypsin Validation Viral Viral Fusion Proteins Viral Pathogenesis Viral Proteins Virion Virus Virus-like particle Work ZIKA analytical method base drug candidate experimental study high throughput technology influenzavirus inhibitor insight instrument iron oxide metal chelator nanosensors neutralizing antibody new technology novel novel strategies overexpression pandemic disease programs rapid detection real time monitoring receptor receptor binding screening single molecule small molecule temporal measurement time use tool undergraduate education undergraduate student virus envelope

项目摘要

Program Director/Principal Investigator: Santra, Santimukul PROJECT SUMMARY/ABSTRACT This application proposes to develop liposome-coated iron oxide nanosensors (LIONs) mimicking as magnetically-labeled host membranes to detect fusion interactions of enveloped viruses. Spin-spin T2 relaxation technique will be established as a generic bioanalytical method to detect dynamic conformational changes in viral glycoproteins that play vital roles in fusion interactions. Within the first aim, influenza fusion protein interactions with LIONs membrane will be studied, considering diverse fusion triggering environmental factors including low pH, protease activation and receptor binding. Further studies to investigate the sensitivity of this LIONs technology in quantifying similar fusion interactions with more native configurations of HA is demonstrated using virus-like particle (VLPs) and shorter domains derived from HA. Similarly, fusion experiments will be carried out using ganglioside-conjugated LIONs. The promising outcome of this research will be screening potential antiviral candidates including small molecules and neutralizing antibodies. The second aim establishes the broad adaptability of this sensitive LIONs technology for evaluation of fusion interactions of enveloped viruses where presence of membrane receptor is an important trigger, for example, SARS-CoV-2. Receptor-conjugated LIONs (R-LIONs) will be developed for the elucidation of this vital process using spike proteins and reporter virus particles (RVPs) of SARS-CoV-2. R-LIONs offers a novel approach of exploring the role of variety of entry receptors that may play an important role in SARS-CoV-2 membrane fusion. The third aim of this proposal determines further advantage of nanosensor technology for the real-time monitoring of virus-mediated cell fusion. SARS-CoV-2 virus entry into ACE2 receptor overexpressing mammalian cells (HEK-293T) will be examined by developing magnetically labeled reporter virus particles (M-RVPs). Parallel experiments will be carried out in the presence of fusion inhibitors and neutralizing antibodies. The important application of this experiment will be demonstrated by screening potential new fusion inhibitors and antiviral candidates. The proposed AREA proposal, if successful, will develop novel nanosensor technology for the rapid detection of fusion interactions of enveloped viruses including SARS-CoV-2 and influenza. This adaptable technology will allow for rapid screening of potential drug candidates and fusion inhibitors of many other enveloped viruses, in a timely fashion. Most importantly, this AREA proposal will initiate the proposed PURE program for undergraduate education, and increase the hands-on biomedical research opportunity and learning for students in our undergraduate-focused institution. OMB No. 0925-0001 and 0925-0002 (Rev. 03/20 Approved Through 02/28/2023) Page Project Summary

项目总监/首席研究员：Santra, Santimukul 项目概要/摘要该申请建议开发脂质体包被的氧化铁纳米传感器（LION），模仿磁性标记的宿主膜来检测包膜病毒的融合相互作用。自旋-自旋 T2 弛豫技术将被建立为通用生物分析方法来检测动态构象变化在融合相互作用中发挥重要作用的病毒糖蛋白。在第一个目标中，流感融合蛋白将研究与 LION 膜的相互作用，考虑到不同的融合触发环境因素包括低 pH、蛋白酶激活和受体结合。进一步研究以调查这种 LIONs 技术在量化与更多原生配置的类似融合相互作用方面的敏感性使用病毒样颗粒 (VLP) 和源自 HA 的较短结构域来证明 HA。同样，融合实验将使用神经节苷脂结合的 LION 进行。这项研究的有希望的结果将正在筛选潜在的抗病毒候选物，包括小分子和中和抗体。第二个目标是建立这种敏感的 LIONs 技术对融合评估的广泛适应性有包膜病毒的相互作用，其中膜受体的存在是一个重要的触发因素，例如， SARS-CoV-2。将开发受体结合的 LION（R-LION）来阐明这一重要过程使用 SARS-CoV-2 的刺突蛋白和报告病毒颗粒 (RVP)。 R-LIONs 提供了一种新颖的方法探索可能在 SARS-CoV-2 膜融合中发挥重要作用的各种进入受体的作用。该提案的第三个目标确定了纳米传感器技术在实时检测方面的进一步优势。监测病毒介导的细胞融合。 SARS-CoV-2 病毒进入 ACE2 受体过表达的哺乳动物将通过开发磁性标记的报告病毒颗粒（M-RVP）来检查细胞（HEK-293T）。平行线实验将在融合抑制剂和中和抗体存在的情况下进行。重要的该实验的应用将通过筛选潜在的新型融合抑制剂和抗病毒药物来证明候选人。拟议的 AREA 提案如果成功，将开发新型纳米传感器技术，用于快速检测包括 SARS-CoV-2 和流感在内的包膜病毒的融合相互作用。这种适应性强的技术将允许快速筛选潜在的候选药物和许多其他包膜病毒的融合抑制剂，及时的时尚。最重要的是，该 AREA 提案将启动拟议的 PURE 计划本科教育，增加学生实践生物医学研究的机会和学习在我们以本科为重点的机构中。 OMB 编号 0925-0001 和 0925-0002（修订版 03/20 已批准至 02/28/2023）页码项目摘要