Cell line and culture conditions

The experimental study employed a serum-free, suspension-adapted HEK293SF-3F6 cell line, derived from human embryonic kidney 293 (HEK293) cells and obtained from the National Research Council of Canada (NRC, Montreal, Canada), kindly provided by Dr. Amine Kamen. The cells were routinely cultured in disposable polycarbonate vented shake flasks (Corning, NY, USA) with volumes of 125 mL, 250 mL, and 1000 mL. The flasks were placed on a Kuhner shaker LT-X (Kuhner, Birsfelden, Switzerland) and incubated at 37 °C, 5% CO2, and 85% relative humidity (RH) and a shaking speed of 130 rpm. To maintain the cells in exponential phase, regular passages were performed every 2–3 days, maintaining cell densities ranging from 0.3 to 0.5 × 106 cells/mL with viabilities above 95%. The culture medium used was HyCell TransFx-H from HyClone (GE Healthcare, Chicago, IL, USA), supplemented with 4 mM GlutaMAX (Gibco, Carlsbad, CA, USA) and 0.1% Pluronic F-68 (Gibco).

Cell concentration and viability were determined using a NucleoCounter NC-3000 automatic cell nuclei counter (Chemometec, Allerod, Denmark), following the manufacturer’s instructions.

Transient transfection and protein expression

Standard transfections were performed at a cell density of 2 × 106 cells/mL and a DNA concentration of 1 µg/mL. The cationic transfection reagent utilized in this study was PEIPro (PolyPlus, Illkirch-Graffenstaden, France). In brief, the appropriate amount of DNA was added to fresh culture medium (10% of the total culture volume to be transfected) and vortexed for 10 s. Then, polyethyleneimine (PEI) was added in a ratio of 2:1 (w/w) with respect to DNA and subjected to vortexing 3 times for 3 s, followed by a 15-min incubation at RT. Finally, the transfection mixture was added to the respective cultures.

Two different plasmids were employed in the experiments: pGag::eGFP (Venereo-Sanchez et al. 2016) and pGag::eGFP-shATM (Díaz-Maneh et al. 2025). Both encoded the HIV-Gag polyprotein fused in-frame with the enhanced green fluorescent protein (eGFP) and contained the same CMV enhancer and CMV promoter. pGag::eGFP-shATM also encoded a short-hairpin RNA (shRNA) sequence under the pU6 promoter targeting the ATM gene. Regarding batch experiments, cell cultures were harvested at 72 h post transfection and centrifuged at 3000 g for 10 min. Supernatants were stored at − 80 °C until use. Perfusion supernatants were generated as previously described (Lavado-García et al. 2020a). Briefly, perfusion was achieved using an alternating tangential flow (ATF) cell retention device (Repligen, Waltham, MA, USA) with 0.2 µm pore size and 0.13 m2 of filtration area hollow fiber modules (Repligen, Waltham, MA, USA) and an ATF flow rate of 0.6 L/min. When performing transfection, perfusion was stopped to incubate the cells with the DNA/PEI solution and reestablished 2 h after transfection. Twenty-four hours after the first transfection, a second transfection was carried out, following the optimized EGE protocol. To carry out media replacement, the filtration rate was set at 0.26 mL/min at the beginning of the process and modified every day depending on the viable cell density to maintain a cell-specific perfusion rate (CSPR) of 30 pL/cell/day.

HIV-1 Gag VLP quantification – fluorimetry

To determine the concentration of HIV-1 Gag::eGFP virus-like particles (VLPs), a validated quantification assay based on fluorimetry was employed (Lavado-García et al. 2021b). The VLP-containing supernatants were obtained by centrifuging at 1000 g for 5 min. GFP intensity, an indicator of VLP concentration, was measured using a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). Relative fluorescence units (RFUs) of transfected samples were calculated by subtracting the readings from the non-transfected control. To convert RFU values into VLP concentration, the following correlation was applied: 

$$VLPs/mL=\:(4.448\times RFU-63.3)\times10^8$$

(1)

HIV-1 Gag VLP quantification – flow virometry (FV)

HIV-1 Gag::eGFP VLPs were quantified utilizing a CytoFLEX LX flow cytometer (Beckman Coulter, Brea, CA, USA). VLPs were detected based on violet side scatter (V-SSC) and FITC fluorescence signals. Laser gains were set to 72 for forward scatter (FSC), 135 for side scatter (SSC), 9 for V-SSC, and 500 for FITC. Prior to analysis, samples were appropriately diluted in filtered PBS to achieve a concentration range of 500 to 5000 events/µL, ensuring an abort rate below 5%. A minimum of 20,000 VLP events were recorded at a flow rate of 10 µL/min. VLPs were distinguished from background noise using V-SSC vs B525-FITC density plots. Results were normalized employing an internal control. Data analysis was conducted using CytExpert v.2.3 software (Beckman Coulter, Brea, CA, USA).

HIV-1 Gag VLP quantification by ELISA

The concentrations of Gag::eGFP polyprotein were determined using a p24 enzyme-linked immunosorbent assay (ELISA) employing the commercially available Innotest® HIV antigen mAb kit (Innogenetics NV, Ghent, Belgium). The assay procedure followed the instructions provided by the manufacturer to ensure accurate results.

HIV-1 Gag VLP and extracellular vesicle quantification – nanoparticle tracking analysis (NTA)

Quantification of both fluorescent and non-fluorescent diffracting particles was performed using Nanoparticle Tracking Analysis (NTA). The measurements were conducted using a NanoSight LM 20 Device (NanoSight Ltd., Amesbury, UK) equipped with a blue laser (488 nm) for quantifying Gag::eGFP VLPs and a neutral density filter for assessing total particle concentration through light scattering. Each value represents the average of three independent measurements. Data analysis was carried out using NanoSight NTA 3.1 software (Malvern Panalytical Ltd., Malvern, UK).

Ultracentrifugation

Concentrated and purified HIV-1 Gag virus-like particles (VLPs) were obtained through a single-cushion ultracentrifugation method. To summarize, a clarified supernatant from a HEK293 cell culture that underwent transient transfection was layered onto a 30% sucrose cushion (3 mL) and centrifuged at 31,000 rpm for 2 h at 4 °C using a SW32 rotor in a Beckman Optima L100XP centrifuge (Beckman Coulter, Brea, CA, USA). Following ultracentrifugation, the supernatant was discarded, and the resulting pellet was resuspended in 1 mL of PBS (GE HealthCare, Chicago, IL, USA). The concentrated material was then stored at − 80 °C for further experimentation.

Click chemistry functionalization of Gag VLPs

Copper-free click chemistry method was utilized to functionalize membrane proteins of purified VLPs coming from ultracentrifugation with Cy5 in a two-step reaction. For primary activation, 5 × 1011 total particles of each sample were incubated for 24 h at 37 °C under constant mixing with 55 µM dibenzocyclooctyne-sulfo-N-hydroxysuccinimidyl ester (DBCO, Sigma Aldrich, St. Louis, MO, USA). Then, 30 µM Cy5-azide (Sigma Aldrich, St. Louis, MO, USA) was added to the mixture and maintained under the same conditions for 12 h. Unconjugated reagent was removed by performing a secondary ultracentrifugation in the conditions abovementioned. Cy5 molecules/VLP were assessed by in-house validated fluorometric assay (García-Trujillo et al. 2025).

Clarification, purification, lyophilization, and HPLC analysis

In short, all cell culture supernatants were clarified with Supracap™ 50V100™ depth filter capsules (Pall Corporation, Port Washington, NY, USA). Each filter was pre-equilibrated with PBS (Hyclone) before filtration. Then, the clarified supernatants were loaded into a 4.7 mL prepacked HiScreen™ CaptoQ™ ImpRes (GE Healthcare, Chicago, IL, USA) automatically operated by an AKTA Pure system (GE Healthcare, Chicago, IL, USA). Both purification protocols have been previously described (Lorenzo et al. 2023). VLP lyophilization was performed according to a previously reported study (González-Domínguez et al. 2021). High-performance liquid chromatography (HPLC) analyses were conducted with an Agilent 1100 Series HPLC System (Agilent Technologies, Santa Clara, CA, USA) equipped with an Agilent AdvanceBio SEC 2.7 µm (Agilent Technologies, Santa Clara, CA, USA) at Servei d’Anàlisi Química (SAQ) of UAB (Barcelona, Spain). Clarified samples were directly plunged provided column equilibration. Recorded signal from PBS was taken as blank.

dsDNA and total protein quantification

The concentration of host cell DNA was determined using the Quant-itTM Picogreen dsDNA assay kit (Sigma Aldrich, San Luis, MO, USA), following the manufacturer’s instructions. In summary, serial dilutions of both the standard and samples were prepared in 1X TE buffer and dispensed into 96-well microplates. Subsequently, 100 µL of the diluted Quant-iTTM PicoGreen® reagent (1:1000 dilution) was added to each well, followed by incubation for 5 min at room temperature. The dsDNA standard curve covered a range of 1.5 to 500 µg/mL, and absorbance readings were taken using the Victor 3 microplate reader (Perkin Elmer, Waltham, MA, USA) before and after the addition of the Quant-iTTM PicoGreen® reagent, as the HIV-1 Gag::eGFP VLPs emit in a similar range. The excitation wavelength was set to 480 nm, and the emission wavelength was set to 520 nm. The DNA concentration of the samples was determined by referencing the standard curve and subtracting the background fluorescence. The concentration of host cell protein was determined using the Micro BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s instructions. To summarize, serial dilutions of both the standard and samples were prepared in phosphate-buffered saline (PBS) and dispensed into the wells of a microplate. Next, 150 µL of the Micro BCA working reagent was added to each well, and the plates were incubated for 1 h at 37 °C. The bovine serum standard curve covered a range of 1.5 to 200 µg/mL, and the absorbance was measured at 562 nm using the Victor 3 microplate reader. The protein concentration of the samples was determined by comparing the absorbance readings to the standard curve.

Dynamic light scattering

The size distribution of nanoparticles was assessed using dynamic light scattering (DLS) with a Zetasizer Nano ZS Instrument (Malvern Instruments, Malvern, UK). The measurements were performed at a temperature of 25 °C and a viscosity of 0.8872 cP. The instrument utilized a He/Ne 633 nm laser and was set at an angle of 173° for data acquisition. Samples were manually introduced into disposable 1 mL cuvettes (Scharlab S.L., Barcelona, Spain) for analysis. Each sample was automatically measured three times to evaluate the measurement error of the instrument.

VLP visualization in cryo-TEM

The morphology and electron density of HIV-1 Gag VLPs were investigated under cryogenic conditions. The samples were rapidly frozen by plunging them into liquid ethane at a temperature of − 180 °C. Approximately 2 µL of the sample was then applied to holey carbon grids that had been pre-treated with glow discharge using a PELCO easiGlow discharger unit (Ted Pella Inc., Redding, CA, USA). The cryo-frozen samples were transferred to a Leica EM GP cryo workstation (Leica Microsystems AG, Wetzlar, Germany) and subsequently examined using a JEM-2011 electron microscope (JEOL Ltd., Tokyo, Japan) operating at an acceleration voltage of 200 kV. Throughout the imaging process, the temperature was maintained at − 180 °C by the continuous addition of liquid ethane. Micrographs were acquired using a CCD-multiscan camera (Gatan Inc., Pleasanton, CA, USA) for further analysis and characterization.

Confocal microscopy imaging

Visualization of VLPs was performed using a LEICA TCS SP8 instrument (Leica Microsystems AG, Weztlar, Germany) equipped with a HyVolution module to enable super resolution imaging. Excitation/emission parameters of each dye used for confocal microscopy were 488 nm/510 nm for GFP and 633/650–795 nm for Cy5. Briefly, 10 µL of each corresponding sample was placed in a glass-mounted slide (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA) and observed under the microscope.

Western blot and SDS-PAGE

For sample preparation, 40 µL of the sample was mixed with 20 µL of 4X LDS (Sigma Aldrich, St. Louis, MO, USA) sample buffer and 7 µL of 2 M dithiothreitol (DTT) (Sigma Aldrich, St. Louis, MO, USA). The mixture was then incubated at 96 °C for 20 min. The prepared samples were stored at 4 °C until the next step. For gel electrophoresis, 20 µL of each sample was loaded onto precast NuPAGE Bis/Tris gels (Invitrogen, Carlsbad, CA, USA) with a concentration range of 4–12%. To monitor low molecular weight, 5 µL of SeeBlue® Plus2 Prestained Protein Standard (Invitrogen, Carlsbad, CA, USA) was included. Gels were run at 200 V and 400 mA for 45 min in MES-SDS running buffer. Coomassie Brilliant Blue G-250-based EZBlueTM Gel Staining Reagent (Sigma Aldrich, St. Louis, MO, USA) was used for protein staining in the SDS-PAGE gels. For western blot analysis, the proteins were transferred onto 0.2 µm nitrocellulose membranes using the Trans-Blot® turbo system (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 5% (w/v) non-fat dry milk in PBS for 30 min, followed by washing with PBS containing 0.1% (w/v) Tween-20. Next, the membranes were incubated overnight at 4 °C with the primary monoclonal antibody against HIV-1 p24 (1:2000) (Icosagen AS, Tartu, Estonia). After washing, immunodetection was performed using an anti-mouse IgG antibody conjugated with a horseradish peroxidase (dilution 1:5000 in PBS 1X) (Bio-Rad Laboratories, Hercules, CA, USA), incubated for 2 h at room temperature, and washed with PBS 0.1% (w/v) Tween-20. Protein bands were visualized by incubating the membranes with a Clarity™ Western ECL Substrate solutions for 2–3 min, and scans were taken in a ChemiDoc MP (Bio-Rad Laboratories, Hercules, CA, USA).

Lipidomic analysesGlycerolipids and neutral lipids

For sample preparation, 750 µL of a chloroform–methanol solution (2:1, v/v) were added along with 0.01% butylated hydroxytoluene (BTH) as a preservative. The solution contained internal standards provided by Avanti Polar Lipids (Croda International, Snaith, UK), including 16:0 D31_18:1 phosphocholine, 16:0 D31_18:1 phosphoethanolamine, 16:0 D31_18:1 phosphoserine, 17:0 lyso-phosphocholine, 17:1 lyso-phosphoethanolamine, 17:1 lyso-phosphoserine, 17:0 D5_17:0 diacylglycerol, 17:0/17:0/17:0 triacylglycerol, and C17:0 cholesteryl ester, each at a concentration of 0.2 nmol. The samples were thoroughly mixed by vortexing and sonication until they achieved a dispersed state. Subsequently, the samples were extracted at a temperature of 48 °C and left to cool overnight. After cooling, the samples were evaporated until dryness and stored at a temperature of − 80 °C until further analysis. Prior to the analysis, 150 µL of methanol was added to the dried samples, followed by centrifugation at 13,000 g for 5 min. The resulting supernatant was then carefully transferred into UPLC vials, ready for injection.

Sphingolipids

To analyze sphingolipid quantities, 750 µL of a methanol-chloroform solution (2:1, v/v) containing specific internal standards, including N-dodecanoylsphingosine, N-dodecanoylglucosylsphingosine, N-dodecanoylsphingosylphosphorylcholine, and C17-sphinganine at a concentration of 0.2 nmol each from Avanti Polar Lipids, was combined with 0.05 mL of serum and the sample of interest. The extraction process was carried out by maintaining the samples at a temperature of 48 °C for an extended period overnight, followed by cooling. Seventy-five microliter of 1 M KOH in methanol was added to the samples, and the entire mixture was incubated at 37 °C for 2 h. After the incubation, 75 µL of 1 M acetic acid was added to the samples to adjust the pH. The samples were then subjected to evaporation until complete dryness, preserving them at − 20 °C until analysis. Prior to it, the dried samples were reconstituted by adding 150 µL of methanol. The reconstituted samples were subjected to centrifugation at a speed of 13,000 g for 5 min to separate the supernatant from any remaining sediment or particles. From the resulting supernatant, 130 µL were carefully transferred to a new vial prepared for injection.

LC-HRMS

LC-HRMS analysis was performed using an Acquity ultra high-performance liquid chromatography (UHPLC) system (Waters, Milford, MA, USA) coupled with a time-of-flight detector (LCT Premier XE). Full scan spectra were acquired from 50 to 1800 Da, with data points collected every 0.2 s. Mass accuracy was maintained at a resolving power of 10,000 using an independent reference spray via the LockSpray interference. Lipid extracts were injected onto an Acquity UHPLC BEH C8 column (1.7 µm particle size, 100 mm × 2.1 mm; Waters, Milford, MA, USA) at a flow rate of 0.3 mL/min and a column temperature of 30 °C.

The mobile phases consisted of methanol with 2 mM ammonium formate and 0.2% formic acid (A) and water with 2 mM ammonium formate and 0.2% formic acid (B). A linear gradient elution program was utilized: 0.0 min: 20% B; 3 min: 10% B; 6 min: 10% B; 15 min: 1% B; 18 min: 1% B; 20 min: 20% B; 22 min: 20% B. Positive identification of compounds was based on accurate mass measurement with an error tolerance of less than 5 ppm and LC retention time compared to standards (92% confidence level). Quantification was performed using the extracted ion chromatogram for each compound with 50 mDa windows. The linear dynamic range was determined by injecting mixtures of internal and natural standards, as indicated above. Sphingolipids: (ceramide, Cer; dihydroceramide, DHCer; sphingomyelin, SM; dihydrosphingomyelin, DHSM; hexosylceramide, HexCer; ceramide Dihexoside, CDH). Glycerophospholipids: (phosphatydilcholine, PC; lyso-phosphatydilcholine, LPC; phosphatydilethanolamine, PE; lyso-phosphatydilethanolamine, LPE). Sterol lipids: (cholesterol, CHOL; cholesteryl ester, CE).

Bioinformatic analyses

Lipid enrichment analyses were performed using LION online software (Molenaar et al. 2019). All lipids were functionally annotated by their relative log2 fold change value compared to the standard Batch pGag::eGFP condition. Then, results were manually annotated and classified regarding lipid origin, lipid class, and functional properties.