Novel approaches to understanding precursor protease autoprocessing in intact viruses

项目摘要 本项目旨在使用两种非常新颖和强大的方法来研究HIV-1蛋白酶(PR)前体 在完整病毒的背景下的二聚化和自动处理。与对成熟公关的研究相比，很少有 了解前体PR是如何被激活和调节的，特别是在生理条件下。我们 开发了一种基于Flow病毒测定法的分析方法，可以对个体进行高通量分析和分类 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。 为了补充这一方法，我们还开发了一种选择性反应监测-质谱法 (SRM-MS)分析，允许同时监测所有Gag和Gag-Pol裂解位点 准确量化裂解部位的加工效率。我们将使用这些检测方法来： 目的1：在完整病毒的背景下确定前体PR自动处理的决定因素。 目标2：确定PR激活的时间与萌芽的关系，并确定过早激活 前体PR活性是受调控的。 目的3：评估耐药性和附属突变如何影响加工、萌发和感染性 在生理条件下的病毒。

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.