A Primer on Proteomic Characterization of Intercellular Communication in a Virus Microenvironment

Abstract

Intercellular communication is fundamental to multicellular life and a core determinant of outcomes during viral infection, where the common goals of virus and host for persistence and replication are generally at odds. Hosts rely on encoded innate and adaptive immune responses to detect and clear viral pathogens, while viruses can exploit or disrupt these pathways and other intercellular communication processes to enhance their spread and promote pathogenesis. While virus-induced signaling can result in systemic changes to the host, striking alterations are observed within the cellular microenvironment directly surrounding a site of infection, termed the virus microenvironment (VME). Mechanisms employed by viruses to condition their VMEs are emerging and are critical for understanding the biology and pathologies of viral infections. Recent advances in experimental approaches, including proteomic methods, have enabled study of the VME in unprecedented detail. In this review article, we provide a primer on proteomic approaches used to study how viral infections alter intercellular communication, highlighting the ways in which these approaches have been implemented and the exciting biology they have uncovered. First, we consider the different molecules secreted by an infected cell, including proteins, either soluble or contained within extracellular vesicles, and metabolites. We further discuss the modalities of interactions facilitated by alteration at the cell surface of infected cells, including immunopeptide presentation and interactions with the extracellular matrix. Finally, we review spatial profiling approaches that have allowed distinguishing how specific subpopulations of cells within a VME respond to infection and alter their protein composition, discussing valuable insights these methods have offered.

Keywords: cell surface; cell-to-cell communication; extracellular matrix; extracellular vesicles; intercellular communication; microenvironment labeling; multiplexed imaging; proteomics; secreted metabolites; secreted proteins; secretome; spatial profiling; surfaceome; viral infection; virus microenvironment.

Graphical abstract

Fig. 1

Infected cells modulate their microenvironment in ways that are unique to the virus and infection site. Many of the intercellular communication strategies employed by infected cells can be directly interrogated using proteomic tools, providing insight into the messages that infected cells relay to the surrounding tissue. Secreted metabolites and proteins, including viral proteins, potentiate diverse responses in recipient cells that have functional consequences for the outcome of infection. Similarly, alterations to protein–protein interactions at the cell surface between cells and cell–ECM can dictate infection outcomes. How cells in a virus microenvironment respond to signaling from infected cells is a function of their spatial proximity to sites of infection. Given this, spatial proteomic profiling methods represent powerful approaches towards uncovering how infection-induced modulation of the microenvironment promotes viral spread and/or pathogenesis.

Fig. 2

Experimental techniques to decipher infection-induced alterations to secreted protein and metabolite profiles.A, measurement of proteins packaged within extracellular vesicles. Extracellular vesicles can be isolated from media conditioned by uninfected or infected cells via (i) density gradient ultracentrifugation, (ii) size-exclusion chromatography, or (iii) immunoaffinity capture prior to proteolytic digestion and LC-MS–based proteomic characterization. B, secreted metabolites can be profiled by enrichment from conditioned media via solvent extraction and subsequent metabolomics by LC-MS. C, quantification of secreted soluble proteins can be achieved by the concentration of protein from conditioned media by ultrafiltration before proteolytic digestion and LC-MS–based proteomic analysis.

Fig. 3

Proteomic tools to probe infection-induced changes to cell surface interactions.A, measurement of proteins expressed on the cell surface. Cell surface proteins can be enriched by (i) chemical labeling of extracellularly facing proteins with an affinity probe or (ii) subcellular fractionation, collecting the plasma membrane fraction, prior to proteolytic digestion and LC-MS–based proteomic characterization. B, detection of antigen peptides presented by MHC molecules can be achieved by immunopurification of MHC molecules prior to LC–MS analysis of antigen peptides released by acidification. C, extracellular matrix proteins can be profiled by removal of cells, followed by solubilization and proteolytic digestion before LC-MS–based proteomics.

Fig. 4

Spatial proteomic approaches to characterize infection-induced alterations to the microenvironment.A, tissue dissection by microscopy-guided laser capture microdissection enables capture of tissue subsections prior to proteolytic digestion and LC-MS–based proteomic characterization. B, spatially resolved antibody-based measurement of proteins in a tissue. Selected proteins can be immunostained using (i) fluorophore- or (ii) DNA oligo-labeled antibodies and detected using fluorescence imaging or, alternatively, using (iii) metal isotype-labeled antibodies with detection by MS. C, unbiased, spatially resolved measurement of proteins, peptides, lipids, or metabolites in a tissue can be achieved by depositing a chemical matrix on a tissue slice prior to raster MALDI-MS. D, deciphering of alterations to proteomes of cells in a virus microenvironment based on spatial proximity to infected cells is enabled by proximity labeling via a secreted and cell penetrable red fluorescent protein followed by cell sorting and MS-based proteomic analysis.