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.

Tryptic peptides unique to MCS proteins were detected and quantified by a Thermo Q Exactive HF, using scheduled runs with 5 or 8 minute isolation windows. Libraries were built with Proteome Discoverer, and PRM data was analyzed in Skyline.

Human fibroblast cells (MRC5) were infected HCMV, HSV-1, and Infl. A and human epithelial cells (RPTE) were infected with HCoV-OC43. Whole-cell lysates were collected at timepoints corresponding to virus entry, genome replication, capsid and envelope assembly, and egress. Tryptic peptides were detected and quantified by a Thermo Q Exactive HF, using scheduled runs with 5 or 8 minute isolation windows. Data for each infection time-course includes biological replicates as follows: HCMV N=6, HSV-1 N=4, Infl. A N=2, HCoV-OC43 N=3.