Formation Of Extracellular Vesicles Research Articles

Proteomic profiling of extracellular vesicles released from vascular smooth muscle cells during initiation of phosphate-induced mineralization

ABSTRACTPurpose/Aim: Elevated serum phosphate is one of the major factors contributing to vascular calcification. Studies suggested that extracellular vesicles released from vascular smooth muscle cells significantly contribute to the initiation and progression of this pathology. Recently, we have demonstrated that elevated phosphate stimulates release of extracellular vesicles from osteogenic cells at the initiation of the mineralization process. Here, we used MOVAS cell line as an in vitro model of vascular calcification to examine whether vascular smooth muscle cells respond to high phosphate levels in a similar way and increase formation of extracellular vesicles. Materials and Methods: Vesicles residing in extracellular matrix as well as vesicles released to culture medium were evaluated by nanoparticle tracking analyses. In addition, using mass spectrometry and protein profiling, protein composition of extracellular vesicles released by MOVAS cells under standard growth conditions and upon exposure to high phosphate was compared. Results: Significant increase of the number of extracellular vesicles was detected after 72 h of exposure of cells to high phosphate. Elevated phosphate levels also affected protein composition of extracellular vesicles released from MOVAS cells. Finally, the comparative analyses of proteins in extracellular vesicles isolated from extracellular matrix and from conditioned medium identified significant differences in protein composition in these two groups of extracellular vesicles. Conclusions: Results of this study demonstrate that exposure of MOVAS cells to high phosphate levels stimulates the release of extracellular vesicles and changes their protein composition.

Rab11 mediates selective recycling and endocytic trafficking in Trypanosoma brucei

Trypanosoma brucei possesses a streamlined secretory system that guarantees efficient delivery to the cell surface of the critical glycosyl-phosphatidylinositol (GPI)-anchored virulence factors, variant surface glycoprotein (VSG) and transferrin receptor (TfR). Both are thought to be constitutively endocytosed and returned to the flagellar pocket via TbRab11+ recycling endosomes. We use conditional knockdown with established reporters to investigate the role of TbRab11 in specific endomembrane trafficking pathways in bloodstream trypanosomes. TbRab11 is essential. Ablation has a modest negative effect on general endocytosis, but does not affect turnover, steady state levels or surface localization of TfR. Nor are biosynthetic delivery to the cell surface and recycling of VSG affected. TbRab11 depletion also causes increased shedding of VSG into the media by formation of nanotubes and extracellular vesicles. In contrast to GPI-anchored cargo, TbRab11 depletion reduces recycling of the transmembrane invariant surface protein, ISG65, leading to increased lysosomal turnover. Thus, TbRab11 plays a critical role in recycling of transmembrane, but not GPI-anchored surface proteins. We proposed a two-step model for VSG turnover involving release of VSG-containing vesicles followed by GPI hydrolysis. Collectively, our results indicate a critical role of TbRab11 in the homeostatic maintenance of the secretory/endocytic system of bloodstream T. brucei.

Isolation, Characterization, and Metal Response of Novel, Acid-Tolerant Penicillium spp. from Extremely Metal-Rich Waters at a Mining Site in Transbaikal (Siberia, Russia).

The role of fungi in metal cycling in acidic environments has been little explored to date. In this study, two acid-tolerant and metal-resistant Penicillium isolates, strains ShG4B and ShG4C, were isolated from a mine site in the Transbaikal area of Siberia (Russia). Waters at the mine site were characterized by extremely high metal concentrations: up to 18gl-1 Fe and > 2gl-1 each of Cu, Zn, Al, and As. Both isolates were identified as Penicillium spp. by phylogenetic analyses and they grew well in Czapek medium acidified to pH 2.5. Resistance to Cu, Cd, Ni, Co, and arsenate was in the range of 1-10gl-1. Further experiments with Penicillium strain ShG4C demonstrated that growth in Cu-containing media was accompanied by the precipitation of Cu-oxalate (moolooite) and the formation of extracellular vesicles enriched in Cu on the mycelia. Vesicles were greatly reduced in size in Cd-containing media and were not formed in the presence of Ni or Co. Cd-oxalate was detected as a crystalline solid phase in Cd-exposed mycelia. Hydrated Ni-sulfate (retgersite) and Co-sulfate (bieberite) were detected in mycelia grown in the presence of Ni and Co, respectively. The results demonstrated that acid-tolerant and metal-resistant Penicillium constitute a component in extremophilic microbiomes, contributing to organic matter breakdown and formation of secondary solid phases at pH ranges found in acid rock drainage.

PS, It's Complicated: The Roles of Phosphatidylserine and Phosphatidylethanolamine in the Pathogenesis of Candida albicans and Other Microbial Pathogens.

The phospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) play important roles in the virulence of Candida albicans and loss of PS synthesis or synthesis of PE from PS (PS decarboxylase) severely compromises virulence in C. albicans in a mouse model of systemic candidiasis. This review discusses synthesis of PE and PS in C. albicans and mechanisms by which these lipids impact virulence in this fungus. This is further compared to how PS and PE synthesis impact virulence in other fungi, parasites and bacteria. Furthermore, the impact of PS asymmetry on virulence and extracellular vesicle formation in several microbes is reviewed. Finally, the potential for PS and PE synthases as drug targets in these various kingdoms is also examined.

The extracellular role of DNA damage repair protein APE1 in regulation of IL-6 expression

The human apurinic/apyrimidinic endonuclease 1 (APE1) is a pleiotropic nuclear protein with roles in DNA base excision repair pathway as well as in regulation of transcription. Recently, the presence of extracellular plasma APE1 was reported in endotoxemic rats. However, the biological significance and the extracellular function of APE1 remain unclear. In this study, we found that monocytes secrete APE1 upon inflammatory challenges. Challenging the monocytic cells with extracellular APE1 resulted in the increased expression and secretion of the pro-inflammatory cytokine IL-6. Additionally, the extracellular APE1 treatment activated the transcription factor NF-κB, followed by its increased occupancy at the IL-6 promoter, resulting in the induction of IL-6 expression. APE1-induced IL-6 further served to elicit autocrine and paracrine cellular responses. Moreover, the extracellular IL-6 promoted the secretion of APE1, thus indicating a functional feedforward loop in this pathway. Furthermore, we show that APE1 is secreted through extracellular vesicles formation via endosomal sorting complex required for transport (ESCRT)-dependent pathway. Together, our study demonstrates a novel role of extracellular APE1 in IL-6-dependent cellular responses.

Extracellular vesicles mediate intercellular communication: Transfer of functionally active microRNAs by microvesicles into phagocytes.

Cell activation and apoptosis lead to the formation of extracellular vesicles (EVs) such as exosomes or microvesicles (MVs). EVs have been shown to modulate immune responses; recently, MVs were described to carry microRNA (miRNA) and a role for MVs in the pathogenesis of autoimmune diseases has been discussed. Here we systematically characterized MVs and exosomes according to their release stimuli. The miRNA content of viable or apoptotic human T lymphocytes and the corresponding MVs was analyzed. miRNA, protein and surface marker expression, as well as cytokine release by human monocytes was measured after EV engulfment. Finally, miRNA expression in T lymphocytes and MVs of healthy individuals was compared with those of systemic lupus erythematosus (SLE) patients. We demonstrate that, depending on the stimuli, distinct subtypes of EVs are released, differing in size and carrying a specific RNA profile. We observed an accumulation of distinct miRNAs in MVs after induction of apoptosis and the transfer of functional miRNA by MVs into human monocytes. MVs released from apoptotic cells provoke less of an inflammatory response than those released from viable cells. MiR-155*, miR-34b and miR-34a levels in T lymphocytes and corresponding MVs were deregulated in SLE when compared to healthy individuals.

The cell biology of the thyroid-disrupting mechanism of dichlorodiphenyltrichloroethane (DDT).

Dichlorodiphenyltrichloroethane (DDT) is an organochlorine known for its pesticide properties and for its negative effects on human health. It was banned in most countries for its toxicity to the endocrine system, but due to its persistence at clinically relevant concentrations in both soil and animal tissues, DDT is still linked to several health and social problems. We have previously shown that DDT exposure is causally related to the extracellular release of vesicular organelles such as microvesicles and/or exosomes by using immunocytochemistry with gold-tagged antibodies and various fluorescent membrane markers. It is now well recognized that microvesicles and/or exosomes organelles are implicated in cell-to-cell communication, and that they are fundamental elements for transferring proteins, RNA, DNA, lipids and transcriptional factors among cells. In this short review, we discussed the role of extracellular vesicle formation in the thyroid-disrupting mechanism of DDT. In particular, we described how DDT, by dislodging the thyrotropin hormone (TSH) receptor from the raft containing compartments of the cells, prevents its activation and internalization. Based on our earlier finding and on the large body of evidence here reviewed, we propose that DDT-induced formation of extracellular vesicles containing the TSH receptor could be directly involved in the development of autoimmune responses against the TSH receptor and that, therefore, their release could lead to the development of the Graves' disease.

Formation and size distribution of self-assembled vesicles

When detergents and phospholipid membranes are dispersed in aqueous solutions, they tend to self-assemble into vesicles of various shapes and sizes by virtue of their hydrophobic and hydrophilic segments. A clearer understanding of such vesiculation processes holds promise for better elucidation of human physiology and disease, and paves the way to improved diagnostics, drug development, and drug delivery. Here we present a detailed analysis of the energetics and thermodynamics of vesiculation by recourse to nonlinear elasticity, taking into account large deformation that may arise during the vesiculation process. The effects of membrane size, spontaneous curvature, and membrane stiffness on vesiculation and vesicle size distribution were investigated, and the critical size for vesicle formation was determined and found to compare favorably with available experimental evidence. Our analysis also showed that the critical membrane size for spontaneous vesiculation was correlated with membrane thickness, and further illustrated how the combined effects of membrane thickness and physical properties influenced the size, shape, and distribution of vesicles. These findings shed light on the formation of physiological extracellular vesicles, such as exosomes. The findings also suggest pathways for manipulating the size, shape, distribution, and physical properties of synthetic vesicles, with potential applications in vesicle physiology, the pathobiology of cancer and other diseases, diagnostics using in vivo liquid biopsy, and drug delivery methods.

Mitochondria-regulated formation of endothelium-derived extracellular vesicles shifts the mediator of flow-induced vasodilation.

To examine the effect of endothelium-derived extracellular vesicles (eEVs) on the mediator of flow-induced dilation (FID), composition, formation, and functional effects on the mediator of FID were examined from two different eEV subtypes, one produced from ceramide, while the other was produced from plasminogen-activator inhibitor 1 (PAI-1). Using video microscopy, we measured internal-diameter changes in response to increases in flow in human adipose resistance arteries acutely exposed (30 min) to eEVs derived from cultured endothelial cells exposed to ceramide or PAI-1. FID was significantly impaired following exposure to 500K/ml (K = 1,000) of ceramide-induced eEVs (Cer-eEVs) but unaffected by 250K/ml. FID was reduced in the presence of PEG-catalase following administration of 250K/ml of Cer-eEVs and PAI-1 eEVs, whereas Nω-nitro-l-arginine methyl ester (l-NAME) had no effect. Pathway analysis following protein composition examination using liquid chromatography tandem mass spectrometry (LC-MS/MS) demonstrated that both subtypes were strongly linked to similar biological functions, primarily, mitochondrial dysfunction. Flow cytometry was used to quantify eEVs in the presence or absence of l-phenylalanine-4'-boronic acid (PBA) and mitochondria-targeted [93-boronophenyl)methyl]triphenyl-phosphonium (mito-PBA), cytosolic and mitochondrial-targeted antioxidants, respectively. eEV formation was significantly and dramatically reduced with mito-PBA treatment. In conclusion, eEVs have a biphasic effect, with higher doses impairing and lower doses shifting the mediator of FID from nitric oxide (NO) to hydrogen peroxide (H2O2). Despite differences in protein content, eEVs may alter vascular function in similar directions, regardless of the stimulus used for their formation. Furthermore, mitochondrial ROS production is required for the generation of these vesicles.NEW & NOTEWORTHY The vascular effect of endothelium-derived extracellular vesicles (eEVs) is biphasic, with higher doses decreasing the magnitude of flow-induced dilation (FID) compared with lower doses that shift the mediator of FID from nitric oxide to H2O2 eEVs may cause vascular dysfunction via similar pathways despite being formed from different stimuli, although both require mitochondrial reactive oxygen species for their formation.

Abstract WP292: Insulin Resistance is Associated with a Reduction of Leukocyte Surface CD36 Expression and an Increase of Soluble CD36

Introduction: Diabetes is a major risk factor for ischemic stroke. Evidence suggests that the deficiency in CD36 is linked to insulin resistance in humans, yet patients with type 2 diabetes often display elevated plasma level of soluble CD36 (sCD36). However, cell type specific expression of CD36 is not well understood in the context of type 2 diabetes. Hypothesis: We hypothesize that insulin resistance is correlated with a reduction in the surface expression of CD36 among inflammatory cells, coincident with an increase in sCD36. Methods: Middle-aged db/db and db/+ mice (30-50 weeks-old) were subjected to left distal middle cerebral artery occlusion (dMCAO) or sham surgery. One, 3, or 7 days after stroke, leukocytes from blood and left hemisphere of the brain were analyzed by flow cytometry for cell type specific expression of CD36. The level of sCD36 in platelet free plasma was determined by ELISA. PPAR-γ agonist pioglitazone was administered at 2.5 mg/kg/day by oral gavage. Oral glucose tolerance test was performed at baseline and following two weeks of drug treatment. Both form of CD36 was also measured in 3-week-old mice without stroke. Results: Infiltrating neutrophils in brain peaked at 1 day after dMCAO, in contrast to a delayed but sustained increase of macrophages from 3 to 7 days post stroke. The adult db/db mice had a lower leukocyte surface expression of CD36 in both blood and brain, but had a higher level of sCD36 compared to age-matched db/+ mice whether at baseline or after stroke. The sCD36 is unlikely to exist in the form of extracellular vesicles (EV) since there were very few CD36(+) EVs detected in either type of mice. Prior to the development of obesity, 3-week-old db/db mice had similar leukocyte CD36 surface expression and plasma level of sCD36 compared to juvenile db/+ mice. Pioglitazone improved glucose sensitivity and enhanced macrophage CD36 surface expression in db/db mice after stroke. Conclusions: The reduced surface expression of CD36 on leukocytes and increased sCD36 was directly correlated with insulin resistance. Ongoing studies will determine the molecular event induced by obesity that triggers the cleavage of CD36 and how it affects macrophage function in the setting of diabetes.

Dichlorodiphenyltrichloroethane (DDT) induced extracellular vesicle formation: a potential role in organochlorine increased risk of Parkinson's disease.

A number of studies have demonstrated that rural living and exposure to pesticides such as dichlorodiphenyltrichloroethane (DDT) highly increase the chances of developing Parkinson's disease. In a previous work, we have found that DDT leads to the formation of vesicular buds that are released from the cells upon fusion of an intermediate endocytic compartment with the plasma membrane. Since extracellular vesicles like exosomes have been implicated in the development of neurodegenerative diseases through the propagation of neurotoxic misfolded proteins from neuron to neuron, in this minireview we propose that organochlorine pesticides could enhance the risk of neurodegenerative diseases by increasing the formation of exosomes.

Designed proteins induce the formation of nanocage-containing extracellular vesicles.

Complex biological processes are often performed by self-organizing nanostructures comprising multiple classes of macromolecules, such as ribosomes (proteins and RNA) or enveloped viruses (proteins, nucleic acids and lipids). Approaches have been developed for designing self-assembling structures consisting of either nucleic acids or proteins, but strategies for engineering hybrid biological materials are only beginning to emerge. Here we describe the design of self-assembling protein nanocages that direct their own release from human cells inside small vesicles in a manner that resembles some viruses. We refer to these hybrid biomaterials as 'enveloped protein nanocages' (EPNs). Robust EPN biogenesis requires protein sequence elements that encode three distinct functions: membrane binding, self-assembly, and recruitment of the endosomal sorting complexes required for transport (ESCRT) machinery. A variety of synthetic proteins with these functional elements induce EPN biogenesis, highlighting the modularity and generality of the design strategy. Biochemical analyses and cryo-electron microscopy reveal that one design, EPN-01, comprises small (~100 nm) vesicles containing multiple protein nanocages that closely match the structure of the designed 60-subunit self-assembling scaffold. EPNs that incorporate the vesicular stomatitis viral glycoprotein can fuse with target cells and deliver their contents, thereby transferring cargoes from one cell to another. These results show how proteins can be programmed to direct the formation of hybrid biological materials that perform complex tasks, and establish EPNs as a class of designed, modular, genetically-encoded nanomaterials that can transfer molecules between cells.

Maternal extracellular vesicles and platelets promote preeclampsia via inflammasome activation in trophoblasts

AbstractPreeclampsia (PE) is a placenta-induced inflammatory disease associated with maternal and fetal morbidity and mortality. The mechanisms underlying PE remain enigmatic and delivery of the placenta is the only known remedy. PE is associated with coagulation and platelet activation and increased extracellular vesicle (EV) formation. However, thrombotic occlusion of the placental vascular bed is rarely observed and the mechanistic relevance of EV and platelet activation remains unknown. Here we show that EVs induce a thromboinflammatory response specifically in the placenta. Following EV injection, activated platelets accumulate particularly within the placental vascular bed. EVs cause adenosine triphosphate (ATP) release from platelets and inflammasome activation within trophoblast cells through purinergic signaling. Inflammasome activation in trophoblast cells triggers a PE-like phenotype, characterized by pregnancy failure, elevated blood pressure, increased plasma soluble fms-like tyrosine kinase 1, and renal dysfunction. Intriguingly, genetic inhibition of inflammasome activation specifically in the placenta, pharmacological inhibition of inflammasome or purinergic signaling, or genetic inhibition of maternal platelet activation abolishes the PE-like phenotype. Inflammasome activation in trophoblast cells of women with preeclampsia corroborates the translational relevance of these findings. These results strongly suggest that EVs cause placental sterile inflammation and PE through activation of maternal platelets and purinergic inflammasome activation in trophoblast cells, uncovering a novel thromboinflammatory mechanism at the maternal-embryonic interface.

Stabilization of membrane necks by adhesive particles, substrate surfaces, and constriction forces.

Membrane remodelling processes involving the formation and fission of small buds require the formation and closure of narrow membrane necks, both for biological membranes and for model membranes such as lipid bilayers. The conditions required for the stability of such necks are well understood in the context of budding of vesicles with bilayer asymmetry and/or intramembrane domains. In many cases, however, the necks form in the presence of an adhesive surface, such as a solid particle or substrate, or the cellular cortex itself. Examples of such processes in biological cells include endocytosis, exocytosis and phagocytosis of solid particles, the formation of extracellular and outer membrane vesicles by eukaryotic and prokaryotic cells, as well as the closure of the cleavage furrow in cytokinesis. Here, we study the interplay of curvature elasticity, membrane-substrate adhesion, and constriction forces to obtain generalized stability conditions for closed necks which we validate by numerical energy minimization. We then explore the consequences of these stability conditions in several experimentally accessible systems such as particle-filled membrane tubes, supported lipid bilayers, giant plasma membrane vesicles, bacterial outer membrane vesicles, and contractile rings around necks. At the end, we introduce an intrinsic engulfment force that directly describes the interplay between curvature elasticity and membrane-substrate adhesion.

Global Proteomic Profiling of the Secretome of Candida albicans ecm33 Cell Wall Mutant Reveals the Involvement of Ecm33 in Sap2 Secretion.

Candida albicans secretes numerous proteins related to cell wall remodeling, adhesion, nutrient acquisition and host interactions. Also, extracellular vesicles containing cytoplasmic proteins are secreted into the medium. The C. albicans ecm33/ecm33 mutant (RML2U) presents an altered cell wall and is avirulent. The proteomic analysis of proteins secreted by RML2U cells identified a total of 170 proteins: 114 and 154 of which correspond to the vesicle-free secretome and extracellular vesicles, respectively. Notably, 98 proteins were common to both samples, and the groups most represented were metabolic and cell wall-related proteins. The results of this study showed that RML2U had an altered pattern of proteins secreted by the classical secretion pathway as well as the formation of extracellular vesicles, including their size, quantity, and protein composition. Specifically, the secretion of aspartic protease 2 (Sap2) was compromised but not its intracellular expression, with bovine serum albumin (BSA) degradation by RML2U being altered when BSA was used as the sole nitrogen source. Furthermore, as recent research links the expression of Sap2 to the TOR (Target Of Rapamycin) signaling pathway, the sensitivity of RML2U to rapamycin (the inhibitor of TOR kinase) was tested and found to be enhanced, connecting Ecm33 with this pathway.

Extracellular vesicles as new pharmacological targets to treat atherosclerosis

Extracellular vesicles released by most cell types, include apoptotic bodies (ABs), microvesicles (MVs) and exosomes. They play a crucial role in physiology and pathology, contributing to “cell-to-cell” communication by modifying the phenotype and the function of target cells. Thus, extracellular vesicles participate in the key processes of atherosclerosis from endothelial dysfunction, vascular wall inflammation to vascular remodeling. The purpose of this review is to summarize recent findings on extracellular vesicle formation, structure, release and clearance. We focus on the deleterious and beneficial effects of extracellular vesicles in the development of atherosclerosis. The potential role of extracellular vesicles as biomarkers and pharmacological targets, their innate therapeutic capacity, or their use for novel drug delivery devices in atherosclerotic cardiovascular diseases will also be discussed.

Tetraspanins in extracellular vesicle formation and function.

Extracellular vesicles (EVs) represent a novel mechanism of intercellular communication as vehicles for intercellular transfer of functional membrane and cytosolic proteins, lipids, and RNAs. Microvesicles, ectosomes, shedding vesicles, microparticles, and exosomes are the most common terms to refer to the different kinds of EVs based on their origin, composition, size, and density. Exosomes have an endosomal origin and are released by many different cell types, participating in different physiological and/or pathological processes. Depending on their origin, they can alter the fate of recipient cells according to the information transferred. In the last two decades, EVs have become the focus of many studies because of their putative use as non-invasive biomarkers and their potential in bioengineering and clinical applications. In order to exploit this ability of EVs many aspects of their biology should be deciphered. Here, we review the mechanisms involved in EV biogenesis, assembly, recruitment of selected proteins, and genetic material as well as the uptake mechanisms by target cells in an effort to understand EV functions and their utility in clinical applications. In these contexts, the role of proteins from the tetraspanin superfamily, which are among the most abundant membrane proteins of EVs, will be highlighted.

Where Do They Come from and Where Do They Go: Candidates for Regulating Extracellular Vesicle Formation in Fungi

In the past few years, extracellular vesicles (EVs) from at least eight fungal species were characterized. EV proteome in four fungal species indicated putative biogenesis pathways and suggested interesting similarities with mammalian exosomes. Moreover, as observed for mammalian exosomes, fungal EVs were demonstrated to be immunologically active. Here we review the seminal and most recent findings related to the production of EVs by fungi. Based on the current literature about secretion of fungal molecules and biogenesis of EVs in eukaryotes, we focus our discussion on a list of cellular proteins with the potential to regulate vesicle biogenesis in the fungi.

Strong antioxidant selenoneine in tuna blood: its roles in selenium redox metabolism

Selenium‐containing imidazole compound, 2‐selenyl‐Nα, Nα, Nα‐ trimethyl‐L‐histidine, “selenoneine” in tuna blood had a strong antioxidant capacity and bound to hemoglobin and myoglobin (Mb), to protect them from iron autoxidation, and reacted with radicals and methylmercury (MeHg). Cell growth of human and zebrafish cultured cells were enhanced in the presence of selenoneine at 5–100 nM, and GPx1 gene expression was induced in dose‐dependent manner. Administration of selenoneine to yellowtail fish reduced ROS and met‐Mb formation, and the meat color change in dark muscle was protected. Selenoneine was incorporated into human embryonic kidney HEK293 transiently overexpressed with organic cations/carnitine transporter‐1 (OCTN1) and zebrafish embryo cells by OCTN1. The Km value of selenoneine uptake was determined to be 13.0 μM in OCTN1‐oeverexpressed HEK293 cells and 9.5 μM in zebrafish erythrocytes, respectively, indicating that selenoneine is the most specific substrate for the OCTN1 in the human and zebrafish cells. When such OCTN1‐expressing cells and embryos were exposed to MeHg–cysteine (MeHgCys), MeHg accumulation was decreased and the excretion and demethylation of MeHg was enhanced by the presence of selenoneine. In addition, exosomal secretion vesicles were detected in the culture water of embryos that had been microinjected with MeHgCys, suggesting that these may be responsible for MeHg excretion and demethylation. In contrast, OCTN1‐deficient embryos accumulated MeHg, and MeHg excretion and demethylation were decreased. Therefore, the selenoneine‐mediated OCTN1 transport system accelerates secretory extracellular lysosomal vesicle formation and thus the demethylation of MeHg.

Extracellular vesicles containing virus-encoded membrane proteins are a byproduct of infection with modified vaccinia virus Ankara

Vaccinia virus is a structurally complex virus that multiplies in the cell cytoplasm. The assembly of Vaccinia virus particles and their egress from infected cells exploit cellular pathways. Most notably, intracellular mature viral particles are enwrapped by Golgi-derived or endosomal vesicles. These enveloped particles, enriched in virus-encoded proteins, migrate to the cell surface where they are released into the extracellular space through fusion of their outer envelope with the cell membrane. We report that baby hamster kidney cells productively infected with the modified vaccinia virus Ankara strain (MVA) also release extracellular vesicles containing virus-encoded envelope proteins but devoid of any virus cargo. Such vesicles were visualized on the cell surface by electron microscopy and immunogold labelling of the B5 envelope protein. A portion of the B5 protein was found to be associated with non-viral material in high speed ultracentrifugation pellets and displayed a buoyant density characteristic of exosomes released by some cell types. An unrelated transmembrane protein (CD40 ligand) encoded by the MVA genome was also incorporated into extracellular vesicles but not into the envelopes that surround extracellular enveloped virus. High speed pellets obtained by centrifugation of culture medium from cells infected with MVA encoding CD40 ligand displayed the ability to induce dendritic cell maturation suggesting that the ligand is on the outer surface of the extracellular vesicles. We propose that the formation of extracellular vesicles after vaccinia virus infection is a byproduct of the pathway leading to the formation of extracellular enveloped virus.