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

呼肠孤病毒-驱动感染的膜相互作用的实时单颗粒分析为了将其基因组物质递送到宿主细胞中进行复制，病毒必须穿过宿主细胞膜。对于缺乏脂质膜的无包膜病毒，成功进入需要宿主膜穿孔。然而，由于缺乏方法来分析短暂发生的进入中的膜相关事件，这一步骤知之甚少。在这个项目中，我们将开发一种新的方法来量化和分析无包膜病毒和宿主膜之间的相互作用，在真实的时间和单粒子分辨率。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初始分解以暴露膜穿透蛋白μ1并产生感染性亚病毒粒子（ISVP）。ISVP经历构象转变以产生ISVP*，并释放肉豆蔻酰化的µ1 N末端片段myr-µ 1 N。myr-µ 1 N的功能对感染至关重要。myr-µ 1 N肽与膜结合，将ISVP招募到膜上，并通过正反馈回路促进额外myr-µ 1 N肽的释放。Myr-µ 1 N也参与孔的形成。myr-µ 1 N如何介导所有这些功能尚不清楚。我们的假设是，ISVP、膜和膜相关myr-µ 1 N之间的进行性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。在目标1中，我们将确定myr-µ 1 N中对膜结合、孔形成和ISVP募集重要的决定簇。我们还将确定破坏这些myr-µ 1 N功能对病毒感染的影响。我们的初步证据表明，与支持的脂质双层孵育的ISVP显示渐进的相互作用-包括随机运动，非随机运动，并最终稳定的连接。在目标2中，使用释放不同量myr-µ 1 N或以不同速率释放myr-µ 1 N的突变病毒，我们将定义myr-µ 1 N释放与粒子运动之间的关系。我们还将确定不断变化的渐进相互作用如何影响病毒感染性。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，开发新的工具来研究这些进入近膜步骤将是揭示其他无包膜病毒的膜渗透事件的变革。

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.