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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV180065	1/1	Homo sapiens	HEL	DG (d)(U)C UF	Deschamps T	2018	50%
Study summary Full title Extracellular Vesicles Released by Herpes Simplex Virus 1-Infected Cells Block Virus Replication in Recipient Cells in a STING-Dependent Manner. All authors Deschamps T, Kalamvoki M Journal J Virol Abstract Herpes simplex virus 1 (HSV-1)-infected cells release extracellular vesicles (EVs) that deliver to u (show more...)Herpes simplex virus 1 (HSV-1)-infected cells release extracellular vesicles (EVs) that deliver to uninfected cells viral factors and host components, such as the stimulator of interferon genes (STING), which activates type I interferon upon foreign DNA sensing. The functions of EVs released by HSV-1-infected cells have remained unknown. Here, we describe a procedure to separate the EVs from HSV-1 virions that is based on an iodixanol/sucrose gradient. STING, along with the EV markers CD63 and CD9, was found in light-density fractions, while HSV components accumulated in heavy-density fractions. HSV-1 infection stimulated the release of EVs from the cells. The EVs derived from infected cells, but not from uninfected cells, activated innate immunity in recipient cells and suppressed viral gene expression and virus replication. Moreover, only the EVs derived from infected cells stimulated the expression of a subset of M1-type markers in recipient macrophages. Conversely, EVs derived from STING-knockdown cells failed to stimulate the expression of these M1-type markers, they activated innate immune responses to a lesser extent in recipient cells, and they did not sustain the inhibition of virus replication. These data suggest that STING from the EV donor cells contributes to the antiviral responses in cells receiving EVs from HSV-1-infected cells. Perturbations in the biogenesis of EVs by silencing CD63 or blocking the activity of the neutral spingomyelinase-2 (nSMase-2) increased the HSV-1 yields. Overall, our data suggest that the EVs released from HSV-1-infected cells negatively impact the infection and could control the dissemination of the virus.IMPORTANCE Extracellular vesicles (EVs) are released by all types of cells as they constitute major mechanism of intercellular communication and have the capacity to alter the functions of recipient cells despite their limited capacity for cargo. How the EVs released by HSV-infected cells could alter the surrounding microenvironment and influence the infection currently remains unknown. The cargo of EVs reflects the physiological state of the cells in which they were produced, so the content of EVs originating from infected cells is expected to be substantially different from that of healthy cells. Our studies indicate that the EVs released by HSV-1-infected cells carry innate immune components such as STING and other host and viral factors; they can activate innate immune responses in recipient cells and inhibit HSV-1 replication. The implication of these data is that the EVs released by HSV-1-infected cells could control HSV-1 dissemination promoting its persistence in the host. (hide) EV-METRIC 50% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin HSV-1 (herpes simplex 1 virus)-infected Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C UF Protein markers EV: TSG101/ CD63/ CD9/ STING/ Flotillin2 non-EV: Proteomics no Show all info Study aim Function/Mechanism of uptake/transfer/Biogenesis/cargo sorting/New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEL EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 11 Lowest density fraction 18% Highest density fraction 6% Total gradient volume, incl. sample (mL) 12 Orientation Top-down Rotor type SW 41 Ti Speed (g) 250000 Duration (min) 120 Fraction volume (mL) 0.5 Fraction processing Dialysis and ultrafiltration Ultra filtration Cut-off size (kDa) 30 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin2/ CD9/ TSG101/ CD63/ STING Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 180 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

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EV-TRACK ID	EV180065
species	Homo sapiens
sample type	Cell culture
cell type	HEL
condition	HSV-1 (herpes simplex 1 virus)-infected
separation protocol	DG (d)(U)C UF
Exp. nr.	1
EV-METRIC %	50