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. 2017 Oct;23(5):713-724.
doi: 10.1007/s13365-017-0552-x. Epub 2017 Jul 31.

Nef Is Secreted in Exosomes From Nef.GFP-expressing and HIV-1-infected Human Astrocytes

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Free PMC article

Nef Is Secreted in Exosomes From Nef.GFP-expressing and HIV-1-infected Human Astrocytes

Pia Pužar Dominkuš et al. J Neurovirol. .
Free PMC article

Abstract

HIV-1 infection of the central nervous system causes HIV-associated neurocognitive disorders, even in aviremic patients. Although astrocyte malfunction was associated to these disorders, their implication is overshadowed by contributions of microglia and macrophages. Astrocytes are infected with HIV-1 in vivo and express a relevant amount of viral protein Nef. Nef was shown to stimulate its own release in exosomes from diverse cell types, which in turn have damaging effects on neighboring cells. Using immunoblotting and electron microscopy, we showed that human astrocytes expressing Nef.GFP similarly release Nef in exosomes. Importantly, Nef.GFP expression increases the secretion of exosomes from human astrocytes up to 5.5-fold, as determined by total protein content and nanoparticle tracking analysis. Protein analysis of exosomes and viruses separated on iodixanol gradient further showed that native or pseudotyped HIV-1-infected human astrocytes release exosomes, which contain Nef. Our results provide the basis for future studies of the damaging role of Nef-exosomes produced by HIV-infected astrocytes on the central nervous system.

Keywords: Astrocytes; Central nervous system; Exosomes; Extracellular vesicles; HIV-1; Nef.

Conflict of interest statement

Conflict of interest The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Nef.GFP-expressing h-astrocytes secrete exosomes containing Nef.GFP. a Immunoblot of cell lysates from Nef.GFP-expressing h-astrocytes collected every 24 h for 3 days post transfection. Antibodies directed against GFP and actin (loading control) were used. CL, cell lysate b, c Protein composition of EVs from Nef.GFP-expressing h-astrocytes cultures, separated on 20–60% sucrose gradient (b), and exosomes (c) isolated from GFP and Nef.GFP-expressing h-astrocytes. Immunoblotting was performed with antibodies directed against GFP, proteins enriched in exosomes (Hsc70, AChE, flotillin, CD63), and against marker for endoplasmic reticulum (calnexin). d TEM image showing exosomes isolated from Nef.GFP-expressing h-astrocytes. The selected exosomes are displayed magnified in the top left corner. Scale bars 100 nm
Fig. 2
Fig. 2
Nef.GFP expression increases the secretion of smaller exosomes from h-astrocytes. a, b Amounts of exosomes released from GFP and Nef.GFP-expressing h-astrocytes were assessed by normalizing the total protein content (a) or the total number of vesicles detected by NTA (b) to the number of transfected cells. c, d Size and concentration of exosomes isolated from GFP-expressing h-astrocytes (c) and Nef.GFP-expressing h-astrocytes (d) assessed by NTA. EXO exosomes
Fig. 3
Fig. 3
HIV-1-infected h-astrocytes release Nef extracellularly. a Immunoblot of pseudotyped NL4-3, YU-2, and NL4-3 Δnef virions. Antibodies directed against viral proteins Gag (p24, p41, and p55), VSV-G, and Nef were used. b Immunoblot of lysed pseudotyped HIV-1-infected astrocytes. Antibodies directed against viral proteins Nef and p24 were used, while tubulin was tested as loading control. c Immunoblot of lysed pseudotyped NL4–3-infected h-astrocytes and the particles they released into the media. Particles were pelleted by ultracentrifugation through sucrose cushion. Antibodies directed against viral proteins (gp120, Nef, p24), proteins enriched in exosomes (Alix, flotillin, AChE), and marker for endoplasmic reticulum (calnexin) and mitochondria (cytochrome c) were used. CL cell lysate, EXO exosomes, VIR virus
Fig. 4
Fig. 4
Pseudotyped HIV-1-infected h-astrocytes secrete exosomes containing Nef. a Schematic illustration of exosomal (AChE+) and viral (p24+) fractions after separation on iodixanol velocity gradient. b AChE activity was quantified by colorimetric assay in all fractions for the presence of exosomes. c The presence of p24 in fractions was quantified by HIV-1 p24 ELISA assay. d Immunoblot of proteins precipitated from 11 collected fractions. Antibodies directed against viral proteins, Nef and p24, and proteins enriched in exosomes, AChE and flotillin, were used. EXO exosomes, VIR virus, AChE acetylcholinesterase
Fig. 5
Fig. 5
HIV-1-infected h-astrocytes secrete exosomes containing Nef. a Immunoblot of native NL4–3 virions. Antibodies directed against viral proteins Gag (p24, p41, and p55), gp120, and Nef were used. b Immunoblot of lysed native NL4-3-infected h-astrocytes. Antibodies directed against viral proteins Nef and Gag were used, while tubulin was tested as loading control. c Immunoblot of proteins precipitated from 11 fractions collected from iodixanol velocity gradient. Antibodies directed against viral proteins, Nef and p24, and proteins enriched in exosomes, AChE and flotillin, were used. EXO exosomes, VIR virus, AChE acetylcholinesterase

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