Membrane contact sites (MCSs) link organelles to coordinate cellular functions across space and time. Although viruses remodel organelles for their replication cycles, MCSs remain largely unexplored during infections. Here, we design a targeted proteomics platform for measuring MCS proteins at all organelles simultaneously and define functional virus-driven MCS alterations by the ancient beta-herpesvirus human cytomegalovirus (HCMV). Integration with super-resolution microscopy and comparisons to herpes simplex virus (HSV-1), Influenza A, and beta-coronavirus HCoV-OC43 infections reveals time-sensitive contact regulation that allows switching anti- to pro-viral organelle functions. We uncover a stabilized mitochondria-ER encapsulation structure (MENC). As HCMV infection progresses, MENCs become the predominant mitochondria-ER contact phenotype and sequentially recruit the tethering partners VAP-B and PTPIP51, supporting virus production. However, premature ER-mitochondria tethering activates STING and interferon response, priming cells against infection. At peroxisomes, ACBD5-mediated ER contacts balance peroxisome proliferation versus membrane expansion, with ACBD5 impacting the titers of each virus tested.

膜接触位点 (MCS) 连接细胞器以协调跨空间和时间的细胞功能。尽管病毒会根据其复制周期重塑细胞器，但在感染过程中，MCS 在很大程度上仍未得到探索。在这里，我们设计了一个靶向蛋白质组学平台，用于同时测量所有细胞器的 MCS 蛋白，并定义了由古老的 β-疱疹病毒人类巨细胞病毒 (HCMV) 引起的功能性病毒驱动的 MCS 改变。与超分辨率显微镜的集成以及与单纯疱疹病毒 (HSV-1)、甲型流感和 β 冠状病毒 HCoV-OC43 感染的比较揭示了时间敏感的接触调节，允许将抗病毒细胞器功能切换为促病毒细胞器功能。我们发现了稳定的线粒体-ER 封装结构 (MENC)。随着 HCMV 感染的进展，MENC 成为主要的线粒体-ER 接触表型，并依次招募系链伙伴 VAP-B 和 PTPIP51，支持病毒生产。然而，过早的 ER-线粒体束缚激活 STING 和干扰素反应，启动细胞抵抗感染。在过氧化物酶体中，ACBD5 介导的 ER 接触平衡过氧化物酶体增殖与膜扩张，ACBD5 影响每种测试病毒的滴度。