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

驱动感染的呼肠孤病毒与膜相互作用的实时单粒子分析为了将其基因组物质传递到宿主细胞中进行复制，病毒必须穿过宿主膜。对于缺乏脂质膜的非包膜病毒，成功进入需要穿透宿主膜。然而，由于缺乏分析瞬时发生的膜相关事件的方法，这一步骤的理解很差。在这个项目中，我们将开发一种新的方法来量化和分析非包膜病毒与宿主膜之间的相互作用，实时和单粒子分辨率。我们将使用呼肠孤病毒作为实验模型。呼肠孤病毒的进入涉及初始分解，暴露膜穿透蛋白µ1，并产生感染性亚病毒粒子（ISVP）。ISVP经过构象转变生成ISVP*s，并释放出微花柱状的µ1 n端片段myr-µ1N。myr-µ1N的功能对感染至关重要。myr-µ1N肽与膜结合，招募isvp到膜上，并通过正反馈回路促进额外myr-µ1N肽的释放。Myr-µ1N也参与了孔隙的形成。myr-µ1N如何介导所有这些功能尚不清楚。我们的假设是ISVPs、膜和膜相关的myr-µ1N之间的三方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Aim 1中，我们将确定myr-µ1N中的决定因素，这些决定因素对膜结合、孔形成和ISVP募集非常重要。我们还将确定破坏这些myr-µ1N功能对病毒感染的影响。我们的初步证据表明，与支持脂质双分子层孵育的isvp表现出渐进的相互作用，包括随机运动、非随机运动和最终的稳定附着。在目标2中，使用释放不同数量的myr-µ1N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。我们还将确定变化的渐进相互作用如何影响病毒传染性。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转折性意义。

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.