SUMMARY The organelles within a eukaryotic host are manipulated by viruses to support successful virus replication and spread of infection, yet the global impact of viral infection on host organelles is poorly understood. Integrating microscopy, sub-cellular fractionation, mass spectrometry, and functional analyses, we conducted a cell-wide study of organelles in primary fibroblasts throughout the timecourse of human cytomegalovirus (HCMV) infection. We used label-free and isobaric-labeling proteomics to characterize nearly 4,000 host and 100 viral proteins, then classified their specific subcellular locations over time using machine learning. We observed a global reorganization of proteins across the secretory pathway, plasma membrane, and mitochondria, including reorganization and processing of lysosomal proteins into distinct subpopulations and translocations of individual proteins between organelles at specific timepoints. We also demonstrate that MYO18A, an unconventional myosin that translocates from the plasma membrane to the viral assembly complex, is necessary for efficient HCMV replication. This study provides a comprehensive resource for understanding host and virus biology during HCMV pathogenesis.

Highlights d Unsupervised clustering revealed subtype with EMT and phosphoprotein signatures d Potential therapeutic vulnerabilities included survivin, NSD3, LSD1, and EZH2 d Rb phosphorylation nominated as a biomarker for trials with CDK4/6 inhibitors d Detailed immune landscape analysis highlighted targetable points of immuneregulation

Organisms are constantly exposed to microbial pathogens in their environments. When a pathogen meets its host, a series of intricate intracellular interactions shape the outcome of the infection. The understanding of these host–pathogen interactions is crucial for the development of treatments and preventive measures against infectious diseases. Over the past decade, proteomic approaches have become prime contributors to the discovery and understanding of host–pathogen interactions that represent anti‐ and pro‐pathogenic cellular responses. Here, we review these proteomic methods and their application to studying viral and bacterial intracellular pathogens. We examine approaches for defining spatial and temporal host–pathogen protein interactions upon infection of a host cell. Further expanding the understanding of proteome organization during an infection, we discuss methods that characterize the regulation of host and pathogen proteomes through alterations in protein abundance, localization, and post‐translational modifications. Finally, we highlight bioinformatic tools available for analyzing such proteomic datasets, as well as novel strategies for integrating proteomics with other omic tools, such as genomics, transcriptomics, and metabolomics, to obtain a systems‐level understanding of infectious diseases.

Summary Viral proteins have evolved to target cellular organelles and usurp their functions for virus replication. Despite the knowledge of these critical functions for several organelles, little is known about peroxisomes during infection. Peroxisomes are primarily metabolic organelles with important functions in lipid metabolism. Here, we discovered that the enveloped viruses human cytomegalovirus (HCMV) and herpes simplex virus (HSV-1) induce the biogenesis of and unique morphological changes to peroxisomes to support their replication. Targeted proteomic quantification revealed a global virus-induced upregulation of peroxisomal proteins. Mathematical modeling and microscopy structural analysis show that infection triggers peroxisome growth and fission, leading to increased peroxisome numbers and irregular disc-like structures. HCMV-induced peroxisome biogenesis increased the phospholipid plasmalogen, thereby enhancing virus production. Peroxisome regulation and dependence were not observed for the non-enveloped adenovirus. Our findings uncover a role of peroxisomes in viral pathogenesis, with likely implications for multiple enveloped viruses.