Real-time single particle analysis of reovirus-membrane interactions that drive infection

实时单颗粒分析呼肠孤病毒-膜相互作用，驱动感染将其基因组材料运送到宿主细胞进行复制，病毒必须穿过宿主膜。对于没有脂膜的无包膜病毒，成功进入需要宿主膜穿孔。然而，由于缺乏分析进入过程中瞬间发生的膜相关事件的方法，对这一步骤了解甚少。在这个项目中，我们将开发一种新的方法来实时和单颗粒分辨率地定量和分析非包膜病毒与宿主膜之间的相互作用。我们将使用呼肠孤病毒作为我们的实验模型。呼肠孤病毒的进入涉及到最初的分解，以暴露膜穿透蛋白µ1并产生传染性亚病毒粒子(ISVP)。ISVP经历构象转换以生成ISVP*S，并释放肉豆蔻化的µ1 N-末端片段MYR-µ1N。MYR-µ1N的功能对感染是必不可少的。MYR-µ1N多肽与膜结合，将ISVP招募到膜上，并通过正反馈循环促进额外MYR-µ1N多肽的释放。MYR-µ1N也参与了孔隙的形成。MYR-µ1N如何调节所有这些功能尚不清楚。我们的假设是，ISVP、膜和膜相关的MYR-µ1N之间的过程三向相互作用是有效感染所必需的。我们将借助两个具体目标来检验这一假设。在目标1中，我们将确定MYR-µ1N中对膜结合、孔形成和ISVP招募至关重要的决定因素。我们还将确定中断这些MYR-µ1N功能中的每一个对病毒感染的影响。我们的初步证据表明，与支持的脂质双层孵育的ISVP表现出渐进的相互作用-包括随机运动、非随机运动和最终稳定的附着。在目标2中，使用释放不同数量MYR-µ1N的突变病毒或以不同速率释放MYR-µ1N的突变病毒，我们将定义MYR-µ1N释放与粒子运动之间的关系。我们还将确定不断变化的相互作用如何影响病毒的传染性。这项工作的完成将阐明非包膜病毒进入的难以捉摸的方面。此外，研究这些膜近端进入步骤的新工具的开发将对揭示其他非包膜病毒的膜穿透事件具有变革性意义。

Real-time single particle analysis of reovirus-membrane interactions that drive infection To deliver their genomic material into the host cells for replication, viruses must cross a host membrane. For non-enveloped viruses that lack a lipid membrane, successful entry requires perforation of the host membrane. However, due the absence of methods to analyze membrane-associated events in entry that occur transiently, this step is poorly understood. In this project, we will develop a new approach to quantify and analyze interactions between non-enveloped viruses and host membranes in real time and at a single particle resolution. We will use reovirus as our experimental model. Entry of reovirus involves initial disassembly to expose the membrane penetration protein µ1 and generate an infectious subvirion particle (ISVP). ISVPs undergo a conformational transition to generate ISVP*s and release a myristylated, µ1 N-terminal fragment, myr-µ1N. The function of myr-µ1N is essential for infection. The myr-µ1N peptide associates with the membrane, recruits ISVPs to the membrane, and promotes the release of additional myr-µ1N peptides via a positive feedback loop. Myr-µ1N is also involved in pore formation. How myr-µ1N mediates all of these functions is unknown. Our hypothesis is that the processive three-way interactions between ISVPs, membrane, and membrane associated myr-µ1N, is required for efficient infection. We will test this hypothesis with the aid of two Specific Aims. In Aim 1, we will identify determinants within myr-µ1N that are important for membrane binding, pore formation, and ISVP recruitment. We will also determine the consequences of disrupting each of these myr-µ1N functions on virus infection. Our preliminary evidence indicates that ISVPs incubated with supported lipid bilayers display progressive interactions - including random motion, non-random motion, and ultimately stable attachment. In Aim 2, using mutant viruses that release different amounts of myr-µ1N or those that release myr-µ1N at different rates, we will define the relationship between myr-µ1N release and particle motion. We will also determine how changing progressive interactions affects virus infectivity. Completion of this work will illuminate the elusive aspects of non-enveloped virus entry. Further, the development of new tools to study these membrane-proximal steps in entry will be transformational for uncovering membrane penetration events of other non-enveloped viruses.