HIV protein Nef expression in human microglia drives the release of distinct Nef-containing extracellular vesicles

Lavrin, Teja (avtor), Loboda, Jure (avtor), Ferdin, Jana (avtor), Levak, Valentina (avtor), Sitar, Simona (avtor), Holcar, Marija (avtor), Resnik, Nataša (avtor), Stenovec, Matjaž (avtor), Trampuš-Bakija, Alenka (avtor), Veranič, Peter (avtor), Žagar, Ema (avtor), Tušek-Žnidarič, Magda (avtor), Pužar Dominkuš, Pia (avtor), Lenassi, Metka (avtor)

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.

• Jezik: Angleški jezik
• Ključne besede: human immunodeficiency virus (HIV), microglia, protein Nef, extracellular vesicles (EVs), HIV-associated neurocognitive disorder (HAND)
• Vrsta gradiva: Članek v reviji
• Tipologija: 1.01 - Izvirni znanstveni članek
• Organizacija: MF - Medicinska fakulteta
• Status publikacije: Objavljeno
• Različica publikacije: Objavljena publikacija
• Leto izida: 2025
• Št. strani: Str. 895-920
• Številčenje: Vol. 6, iss. 4
• PID: 20.500.12556/RUL-179092
• UDK: 616.9
• ISSN pri članku: 2767-6641
• DOI: 10.20517/evcna.2025.106
• COBISS.SI-ID: 259787779
• Datum objave v RUL: 05.02.2026

Gradivo je del revije

• Naslov: Extracellular vesicles and circulating nucleic acids
• Skrajšan naslov: Extracell. vesicles circ. nucleic acids
• Založnik: Alhambra, OAE Publishing
• ISSN: 2767-6641
• COBISS.SI-ID: 175947523

Licence

• Licenca: CC BY 4.0, Creative Commons Priznanje avtorstva 4.0 Mednarodna
• Povezava: http://creativecommons.org/licenses/by/4.0/deed.sl
• Opis: To je standardna licenca Creative Commons, ki daje uporabnikom največ možnosti za nadaljnjo uporabo dela, pri čemer morajo navesti avtorja.

Sekundarni jezik

• Jezik: Slovenski jezik
• Ključne besede: virus humane imunske pomanjkljivosti (HIV), mikroglija, protein Nef, zunajcelični vezikli (EV), z virusom HIV povezana nevrokognitivna motnja (HAND)