Novel approaches to understanding precursor protease autoprocessing in intact viruses

了解完整病毒中前体蛋白酶自动加工的新方法

• 批准号: 10596576
• 负责人: John Christian Tilton
• 金额: $ 51.7万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10596576
• 关键词: Address; Affect; Binding; Biochemical; Bioinformatics; Biological; Biological Assay; CD4 Positive T Lymphocytes; Cell Line; Cells; Complement; Complex; Data; Detection; Dimerization; Dissociation; Drug resistance; Enzymes; Escherichia coli; Event; Fluorescence Resonance Energy Transfer; HIV; HIV-1; HIV-1 protease; Homodimerization; In Vitro; Individual; Integration Host Factors; International; Investigation; Kinetics; Macrophage; Mass Spectrum Analysis; Membrane; Methods; Monitor; Morphologic artifacts; Mutation; Patients; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Physiological; Population; Process; Proteins; Proteomics; Reaction; Reporting; Research Personnel; Resistance; Resolution; Role; Site; Sorting; Time; Transfection; Translations; Viral; Viral Proteins; Virus; design; dimer; experimental study; high throughput analysis; improved; insight; monomer; new therapeutic target; novel; novel strategies; oxidation; particle; pol Gene Products; preference; premature; resistance mutation; stoichiometry; success

Project Abstract This project aims to use two highly novel and powerful assays to investigate HIV-1 protease (PR) precursor dimerization and autoprocessing in the context of intact viruses. In contrast to studies of the mature PR, little is known about how the precursor PR is activated and regulated, particularly under physiological conditions. We have developed a flow virometry-based assay that enables high-throughput analysis and sorting of individual viral particles and provides a rapid and efficient method for identifying determinants of precursor PR activation. To complement this method, we have also developed a selective reaction monitoring-mass spectrometry (SRM-MS) assay that allows simultaneous monitoring of all Gag and Gag-Pol cleavage sites with extremely accurate quantification of cleavage site processing efficiency. We will use these assays to: Aim 1: Identify determinants of precursor PR autoprocessing in the context of intact viruses. Aim 2: Determine the timing of PR activation in relation to budding, and define how premature activation of precursor PR activity is regulated. Aim 3: Evaluate how drug resistance and accessory mutations affect processing, budding, and the infectivity of viruses under physiological conditions.

项目摘要 该项目旨在使用两个高新颖且强大的测定法研究HIV-1蛋白酶（PR）前体 在完整病毒的背景下进行二聚化和自动化。与成熟PR的研究相反，几乎没有 知道如何激活和调节前体PR，特别是在生理条件下。我们 已经开发了基于流病变的测定法，该测定能够进行高通量分析和分类 病毒颗粒，并提供了一种快速有效的方法来识别前体PR激活的决定因素。 为了补充这种方法，我们还开发了一个选择性反应监测质量光谱法 （SRM-MS）测定法，该测定允许同时监视所有GAG和GAG-POL裂解位点，具有极高的 准确量化裂解位点处理效率。我们将使用这些测定法进行： 目标1：在完整病毒的背景下确定前体PR自动加入的决定因素。 AIM 2：确定与萌芽有关的PR激活的时机，并定义如何过早激活 前体PR活性受到调节。 目标3：评估耐药性和配件突变如何影响加工，萌芽和感染力 在生理条件下的病毒。