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.

The complexity of intracellular signaling requires both a diversity of molecular players and the sequestration of activity to unique compartments within the cell. Recent findings on the role of liquid-liquid phase separation provide a distinct mechanism for spatial segregation of proteins to regulate signaling pathway crosstalk. Here we discover that DACT1 is induced by TGF-β and forms protein condensates in the cytoplasm to repress Wnt signaling. These condensates do not localize to any known organelles but rather exist as phase-separated proteinaceous cytoplasmic bodies. Deletion of intrinsically disordered domains within the DACT1 protein eliminates its ability to both form protein condensates and suppress Wnt signaling. Isolation and mass spectrometry analysis of these particles revealed a complex of protein machinery that sequesters Casein Kinase 2, a Wnt pathway activator. We further demonstrate that DACT1 condensates are maintained in vivo and that DACT1 is critical to breast and prostate cancer bone metastasis.

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.

Viral replication in eukaryotes is a process inherently organized in both space and time. Viral components target subcellular organelles to access host machineries required for replication and spread. Diverse viruses are known to alter organelle shape, composition, function, and dynamics as part of their replication cycles. Here, we highlight recent advances in microscopy and proteomic methods that have helped and will continue to help define mechanisms used by viruses to exploit host proteome organization.

Peroxisomes are multifunctional organelles with roles in cellular metabolism, cytotoxicity, and signaling. The plastic nature of these organelles allows them to respond to diverse biological processes, such as virus infections, by remodeling their biogenesis, morphology, and composition to enhance specific functions. During virus infections in humans, peroxisomes act as important immune signaling organelles, aiding the host by orchestrating antiviral signaling. However, more recently it was discovered that peroxisomes can also benefit the virus, facilitating virus–host interactions that rewire peroxisomes to support cellular processes for virus replication and spread. Here, we describe recent studies that uncovered this double-edged character of peroxisomes during infection, highlighting mechanisms that viruses have coevolved to take advantage of peroxisome plasticity. We also provide a perspective for future studies by comparing the established roles of peroxisomes in plant infections and discussing the promise of virology studies as a venue to reveal the uncharted biology of peroxisomes.