20.500.12556/DiRROS-28905 HIV protein Nef expression in human microglia drives the release of distinct Nef-containing extracellular vesicles Aim: Human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND) persist in effectively treated HIV-infected individuals, in part due to HIV reservoirs in brain microglia, which express low levels of viral proteins such as Nef. This study aimed to elucidate how microglia release Nef into the extracellular space, where it exerts its biological functions. Methods: Here, we systematically characterized extracellular particles released from immortalized human microglia (h-microglia) expressing Nef alone or after HIV infection. Importantly, we established a novel h-microglia model harboring a stably integrated Nef tagged with green fluorescent protein (Nef.GFP) transgene under an inducible promoter. Extracellular vesicles (EVs) were enriched from culture media and analyzed for morphology, size, concentration and molecular composition, including Nef content, by (super-resolution) fluorescence microscopy, (immunogold) transmission electron microscopy, asymmetric flow field-flow fractionation coupled to a multi-angle light-scattering detector, nanoparticle tracking analysis, and nano-flow cytometry and immunoblotting. Results: Nef.GFP expression increased particle release up to 11.7-fold compared with controls or known stimulants adenosine triphosphate (ATP) and ionomycin. Compared to the latter, the particles were also significantly smaller (root mean square radius, Rrms = 172 nm) and displayed unique protein and density profiles. All data support the EV nature of the released particles. Approximately half of the Nef.GFP-induced EVs contained Nef (45.5% ± 15.8%), with immunogold labeling confirming its intraluminal localization. Notably, infection with HIV isolates NL4-3 and YU-2 likewise produced Nef-positive EVs distinct from virions. Conclusion: Our findings importantly contribute to understanding the source and characteristics of extracellular Nef in the central nervous system of HIV infected individuals and offer new tools to study HIV Nef biology. Nef-laden EVs should be further investigated as potential therapeutic targets in HAND. human immunodeficiency virus (HIV) microglia protein Nef extracellular vesicles (EVs) HIV-associated neurocognitive disorder (HAND) virus humane imunske pomanjkljivosti (HIV) mikroglija protein Nef zunajcelični vezikli (EV) z virusom HIV povezana nevrokognitivna motnja (HAND) true false true Angleški jezik Slovenski jezik Neznano 2026-04-13 13:38:22 2026-04-13 13:38:22 2026-04-14 03:50:23 0000-00-00 00:00:00 2025 0 0 Nasl. z nasl. zaslona; Opis vira z dne 3. 12. 2025; str. 895-920 iss. 4 Vol. 6 2025 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 616.9 2767-6641 10.20517/evcna.2025.106 259787779 RAZ_Lavrin_Teja_2025.pdf RAZ_Lavrin_Teja_2025.pdf 1 E3EE9E47BB92F2A1C8C53FBD67F4B329 23fe233b69862c4481614d78e988b8dd2be9a9a2a3c9b2c4931a1d3467ff2bec 6472b082-372d-11f1-9cc3-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=43104 https://www.oaepublish.com/articles/evcna.2025.106 1 4b0ce804-372d-11f1-9cc3-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=43103 Univerzitetni klinični center Ljubljana 0 0 0