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

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

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.

• Language: English
• Keywords: human immunodeficiency virus (HIV), microglia, protein Nef, extracellular vesicles (EVs), HIV-associated neurocognitive disorder (HAND)
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: MF - Faculty of Medicine
• Publication status: Published
• Publication version: Version of Record
• Year: 2025
• Number of pages: Str. 895-920
• Numbering: Vol. 6, iss. 4
• PID: 20.500.12556/RUL-179092
• UDC: 616.9
• ISSN on article: 2767-6641
• DOI: 10.20517/evcna.2025.106
• COBISS.SI-ID: 259787779
• Publication date in RUL: 05.02.2026

Record is a part of a journal

• Title: Extracellular vesicles and circulating nucleic acids
• Shortened title: Extracell. vesicles circ. nucleic acids
• Publisher: Alhambra, OAE Publishing
• ISSN: 2767-6641
• COBISS.SI-ID: 175947523

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: virus humane imunske pomanjkljivosti (HIV), mikroglija, protein Nef, zunajcelični vezikli (EV), z virusom HIV povezana nevrokognitivna motnja (HAND)