Dynamic organelle membrane contacts as key regulators of virus replication

项目摘要 细胞器通过直接膜接触形成动态的细胞内网络。膜 接触位点(MCSs)通过蛋白质相互作用连接细胞器膜，是调节的基础 细胞器的结构、组成和动力学。由于这些特征决定了细胞器的功能，MCSs还 控制不同的生物过程，如代谢、运输、细胞器生物发生和细胞凋亡。然而， 在哺乳动物病毒感染期间，细胞器接触在很大程度上仍未被探索，尽管已知 细胞器在广泛的病毒感染中发挥着关键的、往往是保守的作用。此外，许多病毒- 引起细胞器的变化，如囊泡重塑、线粒体碎裂和免疫 调制，都与MCS控制的功能直接相关。我假设膜接触点是 病毒复制的关键调控中心，并在感染期间受到积极调节。我提出了一个综合性的 活细胞成像和定量蛋白质组学方法研究跨亚细胞空间和 感染时间。该工作流程包括靶向蛋白质组学以确定时间MCS蛋白质丰度， 共聚焦显微镜检查细胞器接触表型和基于分子病毒学的功能分析 深入探讨了膜接触部位在病毒复制中的作用机制。我已经去过了 建立了一种高灵敏度跟踪MCS蛋白丰度变化的实验方法 在病毒感染进展过程中的精确度。我进一步将这种方法应用于感染了几个人 病毒，包括疱疹病毒巨细胞病毒(HCMV)和单纯疱疹病毒1型(HSV-1)，以及 事实上，我发现这些病毒会改变MCSs的组成。 感染时间，与每种独特的病毒复制策略相关的时间和细胞器特异性。其中 这些病毒，广泛传播的人类病原体hcmv引起的变化最显著，引发了近 MCS的全球升级和重新布线。鉴于内质网(ER)作为主细胞的已知作用 作为细胞器的调节者，内质网介导的接触将促进病毒复制的关键步骤 周期，我的初步功能分析支持这一点。在这项研究中，我将研究它的功能和 人巨细胞病毒感染中两个关键内质网接触者的调控首先，我将确定ER-Peroxisome接触的作用 在调节病毒组装的过氧酶体可塑性方面。我推测ER-Peroxisome接触增加 在感染过程中，通过病毒与宿主蛋白的相互作用来增强脂质的合成，从而形成 病毒信封。其次，我将调查内质网-内体接触作为囊泡重排的调节因素 病毒粒子组装和出口所必需的，我预测这是以内体类型特定的方式发生来控制的 胆固醇分布和内体转运的方向性。由于MCS是细胞器生物学的基础， 这项工作将有助于扩大对人类病毒复制核心机制的理解。

PROJECT ABSTRACT Cellular organelles form dynamic intracellular networks by engaging in direct membrane contact. Membrane contact sites (MCSs), which link organelle membranes via protein interactions, are fundamental to the regulation of organelle structure, composition, and dynamics. As these features dictate organelle function, MCSs also control diverse biological processes like, metabolism, trafficking, organelle biogenesis, and apoptosis. However, organelle contacts remain largely unexplored during mammalian virus infection despite the knowledge that organelles play critical, and often conserved, roles in a broad range of virus infections. Moreover, many virus- induced changes to organelles, such as vesicular remodeling, mitochondrial fragmentation, and immune modulation, are directly related to MCS-controlled functions. I hypothesize that membrane contact sites are key regulatory hubs of virus replication and actively modulated during infection. I propose an integrative live-cell imaging and quantitative proteomics approach to examine MCS dynamics across subcellular space and infection time. This workflow includes targeted proteomics to determine temporal MCS protein abundances, confocal microscopy to examine organelle contact phenotypes, and molecular virology-based functional assays that delve into the mechanisms underlying the roles of membrane contact sites in virus replication. I have already established an experimental method to track alterations in MCS protein abundances with high sensitivity and precision during the progression of a viral infection. I further applied this method in infections with several human viruses, including the herpesviruses cytomegalovirus (HCMV) and herpes simplex virus 1 (HSV-1), and the orthomyxovirus Influenza A. Indeed, I discovered that these viruses change the composition of MCSs across infection time, with temporal and organelle specificity related to each unique virus replication strategy. Among these viruses, the wide-spread human pathogen HCMV caused the most striking changes, triggering a nearly global upregulation and rewiring of MCSs. Given the known role of the endoplasmic reticulum (ER) as a master regulator of cellular organelles, ER-mediated contacts are poised to facilitate key steps in the virus replication cycle, and my preliminary functional analyses support this. In this study, I will investigate the function and regulation of two critical ER contacts in HCMV infection. First, I will establish the role of ER-peroxisome contacts in modulating peroxisome plasticity for virus assembly. I hypothesize that ER-peroxisome contact is increased during infection and modified by virus-host protein interactions to enhance lipid synthesis for formation of the viral envelope. Second, I will investigate ER-endosome contacts as regulators of the vesicular rearrangements required for virion assembly and egress, which I predict occurs in an endosome type-specific manner to control cholesterol distribution and endosome trafficking directionality. As MCSs are fundamental to organelle biology, this work will help expand the understanding of the mechanisms at the core of human virus replication.