"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”

• 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
• 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

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

项目主任/主要研究者：Santra，Santimukul 项目摘要/摘要 本申请提出开发脂质体包被的氧化铁纳米传感器（LION）， 磁性标记的宿主膜，以检测包膜病毒的融合相互作用。自旋-自旋T2弛豫 将建立一种通用生物分析方法，以检测 在融合相互作用中起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， 这些因素包括低pH、蛋白酶激活和受体结合。进一步研究， 这种LIONs技术在量化与更天然构型的类似融合相互作用方面的灵敏度 使用病毒样颗粒（VLP）和源自HA的较短结构域证明HA。同样， 将使用神经节苷脂缀合的LION进行实验。这项研究的前景将 筛选潜在的抗病毒候选药物，包括小分子和中和抗体。 第二个目标是建立这种敏感的LION技术对融合评估的广泛适应性。 包膜病毒的相互作用，其中膜受体的存在是重要的触发因素，例如， SARS-CoV-2.受体结合的LION（R-LION）将被开发用于阐明这一重要过程 使用SARS-CoV-2的刺突蛋白和报告病毒颗粒（RVP）。R-LION提供了一种新的方法， 探讨SARS-CoV-2膜融合过程中可能起重要作用的各种进入受体的作用。 该建议的第三个目的确定了纳米传感器技术用于实时测量的进一步优势。 监测病毒介导的细胞融合。SARS-CoV-2病毒进入ACE 2受体过表达哺乳动物细胞 将通过显影磁性标记的报告病毒颗粒（M-RVP）检查细胞（HEK-293 T）。平行 实验将在融合抑制剂和中和抗体的存在下进行。重要 该实验的应用将通过筛选潜在的新融合抑制剂和抗病毒药物来证明。 候选人 拟议的区域提案如果成功，将开发新的纳米传感器技术，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。这项适应性强的技术将 允许快速筛选许多其它包膜病毒的潜在候选药物和融合抑制剂， 及时的时尚。最重要的是，该区域提案将启动拟议的PURE计划， 本科教育，并增加学生实践生物医学研究的机会和学习 在我们以本科生为中心的机构。 OMB编号0925-0001和0925-0002（修订版03/20批准至02/28/2023）页码项目摘要