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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, isolation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Isolation protocol
  • Gives a short, non-chronological overview of the different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Details EV-TRACK ID Experiment nr. Species Sample type Isolation protocol First author Year EV-METRIC
EV180065 1/1 Homo sapiens Cell culture supernatant Ultrafiltration
Filtration
(Differential) (ultra)centrifugation
Density gradient
Deschamps T 2018 50%

Study summary

Full title
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% (89th 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Ultrafiltration + Filtration + (Differential) (ultra)centrifugation + Density gradient
Protein markers
EV: None
non-EV: None
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
Sample Condition
HSV-1 (herpes simplex 1 virus)-infected
EV-producing cells
HEL
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Density gradient
Density medium
Iodixanol
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
Detected EV-associated proteins
Flotillin2/ CD9/ TSG101/ CD63/ STING
Fluorescent NTA
Relevant measurements variables specified?
NA
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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