Quantification of organelle membrane contact site protein abundances during infection with human viruses

Viruses rely on organelles to invade, replicate, and spread between host cells. Likewise, organelles underlie the host's ability to sense and respond to pathogen invasion. To subvert cellular functions and turn them to the benefit of virus production and spread, viruses remodel organelle structure and function during infection. We predicted that membrane contact sites (MCSs) underlie virus-driven organelle remodeling events. MCSs use protein interactions to bridge organelle membranes for the direct transfer of biomolecules, coordinating fundamental organelle dynamics. The extent of contact and potential functions of MCSs are dictated by the abundance of MCS-specific proteins at organelle interfaces.

Here, we design a parallel reaction monitoring (PRM) targeted MS assay to quantify MCS protein abundances across time and space. Our assay encompasses nearly all MCS functions and all major cellular organelles (ER, mitochondria, peroxisome, endosomes, lysosomes, autophagosomes, lipid droplets, plasma membrane, Golgi), with 2-5 signature peptides per protein and internal controls. Utilizing MCS-PRM, we uncover temporal- and organelle-specific regulation of MCSs during the infectious cycles of critical human viruses: the beta-herpesvirus human cytomegalovirus (HCMV, also known as HHV-5), herpes simplex virus type 1 (HSV-1), the orthomyxovirus influenza A (Infl. A PR8), and the beta-coronavirus HCoV-OC43.

病毒依赖细胞器完成入侵宿主、自身复制及在宿主细胞间传播的过程。同理，细胞器亦是宿主感知并响应病原体入侵的核心基础。为劫持宿主细胞功能以服务于病毒增殖与传播，病毒会在感染过程中重塑细胞器的结构与功能。我们推测，膜接触位点（membrane contact sites, MCSs）是病毒介导的细胞器重塑事件的核心基础。MCSs通过蛋白质相互作用桥连细胞器膜，实现生物分子的直接转运，进而协调细胞器的核心动态变化。MCSs的接触范围与潜在功能，由细胞器界面处MCS特异性蛋白的丰度所决定。
本研究构建了平行反应监测（parallel reaction monitoring, PRM）靶向质谱（mass spectrometry, MS）分析方法，以时空维度定量检测MCS相关蛋白的丰度。该分析方法覆盖几乎所有MCS功能类型与所有主要细胞细胞器（内质网（endoplasmic reticulum, ER）、线粒体（mitochondria）、过氧化物酶体（peroxisome）、内体（endosomes）、溶酶体（lysosomes）、自噬体（autophagosomes）、脂滴（lipid droplets）、细胞膜（plasma membrane）、高尔基体（Golgi）），每个蛋白对应2-5条特征肽段，并设置内参对照。借助MCS-PRM方法，我们解析了四类重要人类病毒感染周期中MCS的时序特异性与细胞器特异性调控模式：β疱疹病毒科的人类巨细胞病毒（human cytomegalovirus, HCMV，又称HHV-5）、1型单纯疱疹病毒（herpes simplex virus type 1, HSV-1）、正粘病毒科的甲型流感病毒（Infl. A PR8）以及β冠状病毒HCoV-OC43。