Intercellular communication within the virus microenvironment affects the susceptibility of cells to secondary viral infections.

Communication between infected cells and cells in the surrounding tissue is a determinant of viral spread. However, it remains unclear how cells in close or distant proximity to an infected cell respond to primary or secondary infections. We establish a cell-based system to characterize a virus microenvironment, distinguishing infected, neighboring, and distal cells. Cell sorting, microscopy, proteomics, and cell cycle assays allow resolving cellular features and functional consequences of proximity to infection. We show that human cytomegalovirus (HCMV) infection primes neighboring cells for both subsequent HCMV infections and secondary infections with herpes simplex virus 1 and influenza A. Neighboring cells exhibit mitotic arrest, dampened innate immunity, and altered extracellular matrix. Conversely, distal cells are poised to slow viral spread due to enhanced antiviral responses. These findings demonstrate how infection reshapes the microenvironment through intercellular signaling to facilitate spread and how spatial proximity to an infection guides cell fate.

Restructured membrane contacts rewire organelles for human cytomegalovirus infection.

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.

TGF-ß-induced DACT1 biomolecular condensates repress Wnt signalling to promote bone metastasis.

The complexity of intracellular signalling 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 the spatial segregation of proteins to regulate signalling pathway crosstalk. Here, we discover that DACT1 is induced by TGFß and forms protein condensates in the cytoplasm to repress Wnt signalling. These condensates do not localize to any known organelles but, rather, exist as phase-separated proteinaceous cytoplasmic bodies. The deletion of intrinsically disordered domains within the DACT1 protein eliminates its ability to both form protein condensates and suppress Wnt signalling. 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.

Mitochondria and Peroxisome Remodeling across Cytomegalovirus Infection Time Viewed through the Lens of Inter-ViSTA.

Nearly all biological processes rely on the finely tuned coordination of protein interactions across cellular space and time. Accordingly, generating protein interactomes has become routine in biological studies, yet interpreting these datasets remains computationally challenging. Here, we introduce Inter-ViSTA (Interaction Visualization in Space and Time Analysis), a web-based platform that quickly builds animated protein interaction networks and automatically synthesizes information on protein abundances, functions, complexes, and subcellular localizations. Using Inter-ViSTA with proteomics and molecular virology, we define virus-host interactions for the human cytomegalovirus (HCMV) anti-apoptotic protein, pUL37x1. We find that spatiotemporal controlled interactions underlie pUL37x1 functions, facilitating the pro-viral remodeling of mitochondria and peroxisomes during infection. Reciprocal isolations, microscopy, and genetic manipulations further characterize these associations, revealing the interplay between pUL37x1 and the MIB complex, which is critical for mitochondrial integrity. At the peroxisome, we show that pUL37x1 activates PEX11ß to regulate fission, a key aspect of virus assembly and spread.

Peroxisome Plasticity at the Virus-Host Interface.

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.

Location is everything: protein translocations as a viral infection strategy.

Protein movement between different subcellular compartments is an essential aspect of biological processes, including transcriptional and metabolic regulation, and immune and stress responses. As obligate intracellular parasites, viruses are master manipulators of cellular composition and organization. Accumulating evidences have highlighted the importance of infection-induced protein translocations between organelles. Both directional and temporal, these translocation events facilitate localization-dependent protein interactions and changes in protein functions that contribute to either host defense or virus replication. The discovery and characterization of protein movement is technically challenging, given the necessity for sensitive detection and subcellular resolution. Here, we discuss infection-induced translocations of host and viral proteins, and the value of integrating quantitative proteomics with advanced microscopy for understanding the biology of human virus infections.

Infection-Induced Peroxisome Biogenesis Is a Metabolic Strategy for Herpesvirus Replication.

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 type 1 (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.

Exploring and Exploiting Proteome Organization during Viral Infection.

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.