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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV130029	1/1	Homo sapiens	NAY	(d)(U)C	Aqil M	2013	33%
Study summary Full title The HIV-1 Nef protein binds argonaute-2 and functions as a viral suppressor of RNA interference All authors Aqil M, Naqvi AR, Bano AS, Jameel S Journal PLoS One Abstract The HIV-1 accessory protein Nef is an important virulence factor. It associates with cellular membra (show more...)The HIV-1 accessory protein Nef is an important virulence factor. It associates with cellular membranes and modulates the endocytic machinery and signaling pathways. Nef also increases the proliferation of multivesicular bodies (MVBs), which are sites for virus assembly and budding in macrophages. The RNA interference (RNAi) pathway proteins Ago2 and GW182 localize to MVBs, suggesting these to be sites for assembly and turnover of the miRNA-induced silencing complex (miRISC). While RNAi affects HIV replication, it is not clear if the virus encodes a suppressor activity to overcome this innate host response. Here we show that Nef colocalizes with MVBs and binds Ago2 through two highly conserved Glycine-Tryptophan (GW) motifs, mutations in which abolish Nef binding to Ago2 and reduce virus yield and infectivity. Nef also inhibits the slicing activity of Ago2 and disturbs the sorting of GW182 into exosomes resulting in the suppression of miRNA-induced silencing. Thus, besides its other activities, the HIV-1 Nef protein is also proposed to function as a viral suppressor of RNAi (VSR). (hide) EV-METRIC 33% (75th 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 NAY Focus vesicles 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 (d)(U)C Adj. k-factor 253.9 (pelleting) / 253.9 (washing) Protein markers EV: Alix/ CD81/ Beta-actin/ TSG101/ GAPDH non-EV: Cell organelle protein Proteomics no Show all info Study aim Other/HIV-1 accessory protein function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted 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) 60 Pelleting: rotor type SW28 Pelleting: adjusted k-factor 253.9 Wash: volume per pellet (ml) 30 Wash: Rotor Type SW28 Wash: adjusted k-factor 253.9 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD81/ TSG101/ GAPDH/ Beta-actin Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins GAPDH/ Beta-actin

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EV-TRACK ID	EV130029
species	Homo sapiens
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C
Exp. nr.	1
EV-METRIC %	33