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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200015	1/4	Homo sapiens	primary human dermal fibroblasts	DG (d)(U)C Filtration	Streck, Nicholas	2020	89%
Study summary Full title Human Cytomegalovirus Utilizes Extracellular Vesicles To Enhance Virus Spread All authors Nicholas T Streck, Yuanjun Zhao, Jeffrey M Sundstrom, Nicholas J Buchkovich Journal J Virol Abstract Human cytomegalovirus (HCMV) manipulates cellular processes associated with secretory pathways withi (show more...)Human cytomegalovirus (HCMV) manipulates cellular processes associated with secretory pathways within an infected cell to facilitate efficient viral replication. However, little is known about how HCMV infection alters the surrounding cellular environment to promote virus spread to uninfected cells. Extracellular vesicles (EVs) are key signaling molecules that are commonly altered in numerous disease states. Previous reports have shown that viruses commonly alter EVs, which can significantly impact infection. This study finds that HCMV modulates EV biogenesis machinery through upregulation of the endosomal sorting complex required for transport (ESCRT) proteins. This regulation appears to increase the activity of EV biogenesis, since HCMV-infected fibroblasts have increased vesicle release and altered vesicle size compared to EVs from uninfected cells. EVs generated through ESCRT-independent pathways are also beneficial to virus spread in fibroblasts, as treatment with the EV inhibitor GW4869 slowed the efficiency of HCMV spread. Importantly, the transfer of EVs purified from HCMV-infected cells enhanced virus spread. This suggests that HCMV modulates the EV pathway to transfer proviral signals to uninfected cells that prime the cellular environment for incoming infection and enhance the efficiency of virus spread.IMPORTANCE Human cytomegalovirus (HCMV) is a herpesvirus that leads to serious health consequences in neonatal or immunocompromised patients. Clinical management of infection in these at-risk groups remains a serious concern even with approved antiviral therapies available. It is necessary to increase our understanding of the cellular changes that occur during infection and their importance to virus spread. This may help to identify new targets during infection that will lead to the development of novel treatment strategies. Extracellular vesicles (EVs) represent an important method of intercellular communication in the human host. This study finds that HCMV manipulates this pathway to increase the efficiency of virus spread to uninfected cells. This finding defines a new layer of host manipulation induced by HCMV infection that leads to enhanced virus spread. (hide) EV-METRIC 89% (99th 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 Control condition 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 Filtration Protein markers EV: TSG101/ CD81/ CD63 non-EV: Tubulin Proteomics no EV density (g/ml) Density not calculated Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary human dermal fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 130000 Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 5% Highest density fraction 41% Total gradient volume, incl. sample (mL) 5 Sample volume (mL) 1 Orientation Bottom-up Rotor type SW 55 Ti Speed (g) 130000 Duration (min) 960 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 5 Pelleting: duration (min) 120 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 130000 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis PMID previous EV protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63/ TSG101/ CD81 Not detected contaminants Tubulin Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Extra particle analysis NTA Report type Mean Reported size (nm) 170 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 1.01E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field Report type Not Reported EV-concentration No

	EV200015	2/4	Homo sapiens	primary human dermal fibroblasts	DG (d)(U)C Filtration	Streck, Nicholas	2020	89%
Study summary Full title Human Cytomegalovirus Utilizes Extracellular Vesicles To Enhance Virus Spread All authors Nicholas T Streck, Yuanjun Zhao, Jeffrey M Sundstrom, Nicholas J Buchkovich Journal J Virol Abstract Human cytomegalovirus (HCMV) manipulates cellular processes associated with secretory pathways withi (show more...)Human cytomegalovirus (HCMV) manipulates cellular processes associated with secretory pathways within an infected cell to facilitate efficient viral replication. However, little is known about how HCMV infection alters the surrounding cellular environment to promote virus spread to uninfected cells. Extracellular vesicles (EVs) are key signaling molecules that are commonly altered in numerous disease states. Previous reports have shown that viruses commonly alter EVs, which can significantly impact infection. This study finds that HCMV modulates EV biogenesis machinery through upregulation of the endosomal sorting complex required for transport (ESCRT) proteins. This regulation appears to increase the activity of EV biogenesis, since HCMV-infected fibroblasts have increased vesicle release and altered vesicle size compared to EVs from uninfected cells. EVs generated through ESCRT-independent pathways are also beneficial to virus spread in fibroblasts, as treatment with the EV inhibitor GW4869 slowed the efficiency of HCMV spread. Importantly, the transfer of EVs purified from HCMV-infected cells enhanced virus spread. This suggests that HCMV modulates the EV pathway to transfer proviral signals to uninfected cells that prime the cellular environment for incoming infection and enhance the efficiency of virus spread.IMPORTANCE Human cytomegalovirus (HCMV) is a herpesvirus that leads to serious health consequences in neonatal or immunocompromised patients. Clinical management of infection in these at-risk groups remains a serious concern even with approved antiviral therapies available. It is necessary to increase our understanding of the cellular changes that occur during infection and their importance to virus spread. This may help to identify new targets during infection that will lead to the development of novel treatment strategies. Extracellular vesicles (EVs) represent an important method of intercellular communication in the human host. This study finds that HCMV manipulates this pathway to increase the efficiency of virus spread to uninfected cells. This finding defines a new layer of host manipulation induced by HCMV infection that leads to enhanced virus spread. (hide) EV-METRIC 89% (99th 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 HCMV infected 72hpi 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 Filtration Protein markers EV: TSG101/ CD81/ HCMV glycoprotein B/ CD63 non-EV: Tubulin Proteomics no EV density (g/ml) Density not calculated Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary human dermal fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 130000 Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 5% Highest density fraction 41% Total gradient volume, incl. sample (mL) 5 Sample volume (mL) 1 Orientation Bottom-up Rotor type SW 55 Ti Speed (g) 130000 Duration (min) 960 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 5 Pelleting: duration (min) 120 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 130000 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis PMID previous EV protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63/ HCMV glycoprotein B/ TSG101/ CD81 Not detected contaminants Tubulin Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Extra particle analysis NTA Report type Mean Reported size (nm) 158 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 3.51E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field Report type Not Reported EV-concentration No

	EV200015	3/4	Homo sapiens	primary human dermal fibroblasts	DG (d)(U)C Filtration	Streck, Nicholas	2020	44%
Study summary Full title Human Cytomegalovirus Utilizes Extracellular Vesicles To Enhance Virus Spread All authors Nicholas T Streck, Yuanjun Zhao, Jeffrey M Sundstrom, Nicholas J Buchkovich Journal J Virol Abstract Human cytomegalovirus (HCMV) manipulates cellular processes associated with secretory pathways withi (show more...)Human cytomegalovirus (HCMV) manipulates cellular processes associated with secretory pathways within an infected cell to facilitate efficient viral replication. However, little is known about how HCMV infection alters the surrounding cellular environment to promote virus spread to uninfected cells. Extracellular vesicles (EVs) are key signaling molecules that are commonly altered in numerous disease states. Previous reports have shown that viruses commonly alter EVs, which can significantly impact infection. This study finds that HCMV modulates EV biogenesis machinery through upregulation of the endosomal sorting complex required for transport (ESCRT) proteins. This regulation appears to increase the activity of EV biogenesis, since HCMV-infected fibroblasts have increased vesicle release and altered vesicle size compared to EVs from uninfected cells. EVs generated through ESCRT-independent pathways are also beneficial to virus spread in fibroblasts, as treatment with the EV inhibitor GW4869 slowed the efficiency of HCMV spread. Importantly, the transfer of EVs purified from HCMV-infected cells enhanced virus spread. This suggests that HCMV modulates the EV pathway to transfer proviral signals to uninfected cells that prime the cellular environment for incoming infection and enhance the efficiency of virus spread.IMPORTANCE Human cytomegalovirus (HCMV) is a herpesvirus that leads to serious health consequences in neonatal or immunocompromised patients. Clinical management of infection in these at-risk groups remains a serious concern even with approved antiviral therapies available. It is necessary to increase our understanding of the cellular changes that occur during infection and their importance to virus spread. This may help to identify new targets during infection that will lead to the development of novel treatment strategies. Extracellular vesicles (EVs) represent an important method of intercellular communication in the human host. This study finds that HCMV manipulates this pathway to increase the efficiency of virus spread to uninfected cells. This finding defines a new layer of host manipulation induced by HCMV infection that leads to enhanced virus spread. (hide) EV-METRIC 44% (84th 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 HCMV infected 72hpi DMSO-treated 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 Filtration Protein markers EV: CD63 non-EV: None Proteomics no EV density (g/ml) Density not calculated Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary human dermal fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 130000 Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 5% Highest density fraction 41% Total gradient volume, incl. sample (mL) 5 Sample volume (mL) 1 Orientation Bottom-up Rotor type SW 55 Ti Speed (g) 130000 Duration (min) 960 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 5 Pelleting: duration (min) 120 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 130000 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis PMID previous EV protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63 Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Extra particle analysis NTA Report type Not Reported Particle yield NA NA Report type Not Reported EV-concentration No

	EV200015	4/4	Homo sapiens	primary human dermal fibroblasts	DG (d)(U)C Filtration	Streck, Nicholas	2020	44%
Study summary Full title Human Cytomegalovirus Utilizes Extracellular Vesicles To Enhance Virus Spread All authors Nicholas T Streck, Yuanjun Zhao, Jeffrey M Sundstrom, Nicholas J Buchkovich Journal J Virol Abstract Human cytomegalovirus (HCMV) manipulates cellular processes associated with secretory pathways withi (show more...)Human cytomegalovirus (HCMV) manipulates cellular processes associated with secretory pathways within an infected cell to facilitate efficient viral replication. However, little is known about how HCMV infection alters the surrounding cellular environment to promote virus spread to uninfected cells. Extracellular vesicles (EVs) are key signaling molecules that are commonly altered in numerous disease states. Previous reports have shown that viruses commonly alter EVs, which can significantly impact infection. This study finds that HCMV modulates EV biogenesis machinery through upregulation of the endosomal sorting complex required for transport (ESCRT) proteins. This regulation appears to increase the activity of EV biogenesis, since HCMV-infected fibroblasts have increased vesicle release and altered vesicle size compared to EVs from uninfected cells. EVs generated through ESCRT-independent pathways are also beneficial to virus spread in fibroblasts, as treatment with the EV inhibitor GW4869 slowed the efficiency of HCMV spread. Importantly, the transfer of EVs purified from HCMV-infected cells enhanced virus spread. This suggests that HCMV modulates the EV pathway to transfer proviral signals to uninfected cells that prime the cellular environment for incoming infection and enhance the efficiency of virus spread.IMPORTANCE Human cytomegalovirus (HCMV) is a herpesvirus that leads to serious health consequences in neonatal or immunocompromised patients. Clinical management of infection in these at-risk groups remains a serious concern even with approved antiviral therapies available. It is necessary to increase our understanding of the cellular changes that occur during infection and their importance to virus spread. This may help to identify new targets during infection that will lead to the development of novel treatment strategies. Extracellular vesicles (EVs) represent an important method of intercellular communication in the human host. This study finds that HCMV manipulates this pathway to increase the efficiency of virus spread to uninfected cells. This finding defines a new layer of host manipulation induced by HCMV infection that leads to enhanced virus spread. (hide) EV-METRIC 44% (84th 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 HCMV infected 72hpi GW4869-treated 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 Filtration Protein markers EV: CD63 non-EV: None Proteomics no EV density (g/ml) Density not calculated Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary human dermal fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 130000 Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 5% Highest density fraction 41% Total gradient volume, incl. sample (mL) 5 Sample volume (mL) 1 Orientation Bottom-up Rotor type SW 55 Ti Speed (g) 130000 Duration (min) 960 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 5 Pelleting: duration (min) 120 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 130000 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis PMID previous EV protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63 Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Extra particle analysis NTA Report type Not Reported EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample Not reported Report type Not Reported EV-concentration No

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EV-TRACK ID	EV200015
species	Homo sapiens
sample type	Cell culture
cell type	primary human dermal fibroblasts
condition	Control condition	HCMV infected 72hpi	HCMV infected 72hpi DMSO-treated	HCMV infected 72hpi GW4869-treated
separation protocol	DG (d)(U)C Filtration	DG (d)(U)C Filtration	DG (d)(U)C Filtration	DG (d)(U)C Filtration
Exp. nr.	1	2	3	4
EV-METRIC %	89	89	44	44