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. 2024 Jul;13(7):e12478.
doi: 10.1002/jev2.12478.

HIV-1 Nef is carried on the surface of extracellular vesicles

Christophe Vanpouille  1 , Beda Brichacek  2 , Tatiana Pushkarsky  2 , Larisa Dubrovsky  2 , Wendy Fitzgerald  1 , Nigora Mukhamedova  3 , Sofia Garcia-Hernandez  4 , Doreen Matthies  5 , Anastas Popratiloff  4 , Dmitri Sviridov  3   6 , Leonid Margolis  1   7 , Michael Bukrinsky  2
Affiliations

HIV-1 Nef is carried on the surface of extracellular vesicles

Christophe Vanpouille et al. J Extracell Vesicles. 2024 Jul.

Abstract

Extracellular vesicles (EVs) serve as pivotal mediators of intercellular communication in both health and disease, delivering biologically active molecules from vesicle-producing cells to recipient cells. In the context of HIV infection, EVs have been shown to carry the viral protein Nef, a key pathogenic factor associated with HIV-related co-morbidities. Despite this recognition, the specific localisation of Nef within the vesicles has remained elusive. This study addresses this critical knowledge gap by investigating Nef-containing EVs. Less than 1% of the total released Nef was associated with EVs; most Nef existed as free protein released by damaged cells. Nevertheless, activity of EV-associated Nef in downregulating the major cholesterol transporter ABCA1, a critical aspect linked to the pathogenic effects of Nef, was comparable to that of free Nef present in the supernatant. Through a series of biochemical and microscopic assays, we demonstrate that the majority of EV-associated Nef molecules are localised on the external surface of the vesicles. This distinctive distribution prompts the consideration of Nef-containing EVs as potential targets for immunotherapeutic interventions aimed at preventing or treating HIV-associated co-morbidities. In conclusion, our results shed light on the localisation and functional activity of Nef within EVs, providing valuable insights for the development of targeted immunotherapies to mitigate the impact of HIV-associated co-morbidities.

Keywords: ELISA; HIV‐1; Nef; extracellular vesicles.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Characterisation of Nef EVs. (a) Nef EVs, obtained through ultracentrifugation from Nef‐transfected HEK293T cells, underwent fractionation using iodixanol gradient. 13 fractions were collected, and Nef concentrations (pg/mL) along with particle concentrations (particles/mL) for the first twelve fractions were determined through Nef ELISA and nanoparticle tracking analysis, respectively. (b) Fractionated on the iodixanol gradient Nef EVs were subjected to Western blot analysis for the presence of Alix, CD63, TSG101, Hsp70, Nef, CD9 and CD81. (c) Nef EVs were analysed utilising the ExoView platform. Particle capture was achieved using anti‐CD63, anti‐CD81, or anti‐CD9 antibodies, followed by detection with fluorescently labelled anti‐CD63‐CF647 (red), CD81‐CF555 (green) or CD9‐CF488 (blue). Anti‐IgG served as a capture control. The graph depicts means with standard errors of the means (SEM) for the number of EVs positive for CD63 (red), CD81 (green) and CD9 (blue) from three capture spots in two independent measurements.
FIGURE 2
FIGURE 2
Analysis of ABCA1 abundance in EV‐treated cells. (a) HeLa‐ABCA1 cells were subjected to treatment with 0.9 × 1010 EVs/mL of control or Nef EVs (45.8 pg/mL of Nef) (left panel). Another set of cells was treated with ultrasupernatants remaining after the precipitation of control EVs (Cont US) or Nef EVs (Nef US) (right panel). Nef US contained 25 ng/mL of Nef. Analysis of ABCA1 was conducted on ProteinSimple Jess instrument, with total protein serving as the loading control. Lanes represent technical replicates. (b) Quantification of results in panel A was performed using ProteinSimple software. p‐Values, obtained through unpaired t‐test, are displayed above the corresponding bars.
FIGURE 3
FIGURE 3
Nef localisation on the EV surface. (a) Nef EVs, obtained from HEK293T cells transfected with the Nef vector, were divided into two equal volumes and treated with proteinase K. The quantification of Nef associated with Nef EVs in the presence or absence of 0.5% Triton X‐100 was performed using ELISA. One representative experiment from two performed is presented. (b) The amount of syntenin associated with Nef EVs in the presence of 0.5% Triton X‐100 was quantified using ELISA. One representative experiment from two is shown. (c) Triplicate samples of EVs produced by Nef‐transfected SupT1 cells were treated or not with proteinase K, and analysed by Western blot for Nef and internal protein Alix. (d) Quantification of the bands in panel C was conducted using an ImageJ application, with presented results illustrating the Nef/Alix ratio. Statistical analysis was carried out by unpaired Student's t test, and the p‐value is displayed above the sample values. (e) EVs isolated from the conditioned media of HIVADA‐infected or uninfected MDM cell cultures, as well as EVs from the conditioned media of HEK293T cells transfected with Nef‐expressing (Nef EVs) or empty vector (Cont EVs), were equalised based on the protein content and applied to an ELISA plate coated with the anti‐Nef monoclonal antibody. The signal was revealed using the anti‐CD63 polyclonal antibody, followed by an HRP‐conjugated secondary antibody. The bars in the graph represent the mean ± SEM of the OD450 ‐ OD540 difference, with individual points indicating technical replicates. Statistical analysis was performed using ordinary one‐way ANOVA with Sidak correction for multiple comparisons. (f) Bodipy‐labelled EVs were captured by magnetic nanoparticles coupled with anti‐tetraspanin antibodies (anti‐CD63, anti‐CD81 and anti‐CD9), and the EV surface was stained for the presence of Nef with AF647‐labelled anti‐Nef antibody (left panel). Staining of the surface Nef, along with intravesicular Nef staining, was performed with the same AF647‐labelled anti‐Nef‐ antibody after fixation and permeabilisation (right panel). A representative experiment of two is shown. (g, h) Control EVs and Nef‐RFP EVs, isolated from supernatants of transfected HEK293T cells, were analysed using two anti‐RFP antibodies, which were alternately utilised for capturing and detecting.
FIGURE 4
FIGURE 4
Immunogold labelling of Nef EVs. Representative electron microscopy (EM) images of immunogold‐labelled EVs using the anti‐Nef antibody are presented. The EVs exhibit a cup‐shaped morphology. The insets show selected EVs at higher magnification. Small black arrows indicate background gold particles bound to the grids. Scale bars are 100 nm. (a) A group of EVs of various sizes, with one being immunogold positive (large black arrow) whilst the remaining EVs are negative (large white arrows). (b) EVs positively labelled with the anti‐Nef antibody (large black arrows). (c) Control EVs from cells transfected with an empty vector, displaying negativity for the anti‐Nef antibody (large white arrows). (d) Quantification of Nef‐positive immunogold‐labelled EVs from three tiled images from two independent experiments. Results are presented as mean ± SEM.
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