Cell and cell culture
Vero E6 cells and Hela cells were obtained from the National Collection of Authenticated Cell Cultures, China. These cells were cultured in 90% DMEM basal medium (Gibco, USA), supplemented with 10% fetal bovine serum (Gibco, USA) and 100 units/mL penicillin/streptomycin (Gibco, USA), and maintained at 37 °C in a 5% CO2 atmosphere.
Plasmid construction and transfection
Sequences of the wild-type SARS-CoV-2 envelope protein (2-E) were synthesized by the Beijing Genomics Institute (BGI, China). The 2-E-coding sequence, tagged with HA or mCherry, was cloned into the pcDNA3.1 plasmid for 2-E expression. Transfections were conducted using Lipofectamine 3000 (Invitrogen, USA) following the manufacturer’s protocol. The cell culture medium was replaced 6 h after transfection.
Preparation of 2-E-EVs
Vero E6 cells were seeded at 5 × 104 cells per well in a six-well plate and transfected with 2-E-mCherry plasmids the next day. After 24 h of transfection, the medium supernatant was collected and subjected to gradient centrifugation: 100 g for 10 min, 200 g for 10 min, and 400 g for 20 min. The precipitate was resuspended in PBS (Meilunbio, China) and filtered by a microporous filter (5 μm, 8 μm, and 10 μm) (Meck, Germany). The filtered sample was then collected for subsequent experiments.
Transmission electron microscopy (TEM)
The method for preparing 2-E-EVs, as described above, was followed by embedding the centrifuge precipitate in Matrigel (Yeasen, China). The preparation and imaging conditions for the electron microscope samples were consistent with those previously described [4].
Preparation of exosomes and nanoparticle tracking analysis (NTA)
Vero E6 cells were cultured in a serum-free medium for 72 h. The supernatant was then centrifuged at 10,000 × g, 4 °C, for 45 min, followed by filtration through a 0.45 µm microporous filter, and further centrifuged at 100,000 × g, 4 °C, for 70 min. The supernatant was discarded, and the precipitate was resuspended, centrifuged again under the same conditions, and finally resuspended for further experiments.
The sample particle size was measured using nanoparticle tracking analysis (ZetaVIEW, Particle Metrix, Germany). Samples were diluted by 1× PBS buffer (MA0015, Meilunbio, Dalian, China) to measure particle size and concentration. The 110 nm polystyrene particles were used to calibrate the ZetaView system. Date was further analyzed by the software ZetaView (version 8.05.14 SP7).
Untargeted lipidomics analysis
Vero E6 cells, transfected with 2-E-mCherry plasmids or pcDNA3.1-vector for 24 h, were used to collect 2-E-EVs and exosomes. The cells, both 2-E-transfected and vector-transfected, were treated with trypsin, collected by centrifugation (800 rpm, 5 min), and resuspended in 300 µL MeOH/H2O buffer, vortexed for 30 s. Subsequently, 600 µL MTBE buffer was added, vortexed for 30 s, and the mixture was centrifuged at 14,000 × g, 4 °C, for 10 min. The upper supernatant was collected and freeze-dried at –80 °C.
UPLC-MS analysis was performed using an HPLC Vanquish (Thermo Fisher) with an Acclaim C30 liquid chromatographic column (2.1 × 100 mm). The mobile phase consisted of solvent A (10 mM Ammonium formate, Acetonitrile: H2O = 6:4, 0.1% Formic acid) and solvent B (10 mM Ammonium formate, Isopropanol: Acetonitrile = 9:1, 0.1% Formic acid) Afterwards, samples were analyzed by Q Exactive HF-X (Thermo, USA) in both Positive and Negative ion modes, Mass spectrometry data was analyzed using Progenesis-QI software.
Proteomics analysis.
Vero E6 cells transfected with 2-E plasmids and EVs were lysed in SDT buffer (4% SDS, 100 mM DTT, 100 mM Tris-HCl, pH 7.6). Protein digestion was conducted using the Filter-Aided Sample Preparation (FASP) procedure. Digestion was performed overnight at 37 °C with trypsin (Promega, USA) at a 1:50 (w/w) concentration in 50 mM NH4HCO3. Following digestion, peptides were eluted via centrifugation, subsequently dried using vacuum centrifugation, and desalted through C18 solid-phase extraction. NanoDrop 2000 (Thermo Scientific, USA) was employed to ascertain peptide concentration.
Analysis of peptides was conducted using a nanoElute nanoflow high-pressure liquid chromatography (HPLC) system (Bruker Daltonics, USA), linked to a hybrid trapped ion mobility spectrometry (TIMS) quadrupole TOF mass spectrometer (Bruker Daltonics, USA) via a CaptiveSpray nano-electrospray ion source. Data acquisition in diaPASEF mode on the timsTOF Pro was followed by analysis with DIA-NN (version 1.7.16), employing specific parameters: Trypsin/P as Protease, a maximum of one missed cleavage, N-term methionine excision, C carbamidomethylation as fixed modifications, a peptide length range of 7–30, a precursor m/z range of 300–1800, and a fragment m/z range of 200–1800.
DIA-NN output files underwent further analysis and visualization in R statistical programming language (version 3.5.3). Protein abundance data from Vero E6 cells were normalized using the median centering method, selecting only proteins quantified across all samples. Student’s t-test was utilized to identify differentially expressed proteins in the 2-E versus Vector group. Over-representation analysis with Gene Ontology, KEGG, and Reactome pathway gene sets was conducted via WebGestalt.
Protein‒lipid correlation network
To build a network, protein-lipid pairs (Golgi-related proteins and upregulated lipids in 2-E-EVs) with strong correlations (r > |0.5|. p-value < 0.05) were used. Protein-lipid correlation network was conducted by custom R. The network diagram was produced by the Cytoscape software [16].
Confocal fluorescence microscopy
Confocal fluorescence microscopy studies were executed using the Leica TCS-SP8 STED system (Leica Microsystems, Germany). Vero E6 cells were seeded at 3 × 104 per well in 15 mm glass-covered dishes (NEST, China) and co-transfected the following day with 2-E, Golgi-mCherry (B4GALT1-mCherry), and plasma-EGFP (GAP43-EGFP). Six hours post-transfection, the culture medium was replaced. For dual or triple color imaging, multitracking was utilized. Cells were maintained in a living cell station during confocal studies. Quantitative confocal image analyses were performed using Image J software. Parameters such as the number, area, and circularity of Golgi apparatus particles were quantified via the Analyze Particle plugin in Image J [17]. Otsu’s thresholding algorithm was applied for binary image conversion, with a circularity value of 1, indicating a perfect circle. Pearson’s correlation coefficient was calculated by the Colocalization plugin in Image J. The Live images showcasing 2-E-EVs production and Golgi fragmentation were recorded using the Leica TCS-SP8 STED system (Leica Microsystems, Germany) with a 100× oil objective.
Western blot
For Western blot analyses, samples were lysed with IP lysis buffer (Beyotime, China), and protein content was quantified using a BCA protein assay kit (Thermo, USA) according to the manufacturer’s instructions. Equivalent protein amounts were loaded per sample and separated by SDS-PAGE. After transfer to PVDF membranes, the membranes were incubated with blocking buffer and subsequently with antibodies against TSG101 (Proteintech, USA), Flotillin-1 (CST, USA), CD9 (CST, USA), CD63 (ABconal, USA), CD81 (Proteintech, USA), HSP70 (CST, 4876), NDST1 (ABconal, USA), PIGK (Proteintech, USA), CPQ (ABconal, USA), Annexin V(CST, USA), Intergrin-β1 (ABconal, USA) in EV marker western blot, and GAPDH (ABconal, USA), HA (CST, USA), Caspase-3 (CST, USA), Cleaved caspase-3 (CST, USA), GRASP65 (ABconal, USA), P115 (ABconal, USA), GM130(CST, USA) in Golgi fragmentation western blot overnight at 4 °C. After thrice washing with TBST buffer, the membranes were incubated with horseradish peroxidase-labeled goat anti-rabbit/mouse IgG secondary antibody (Yeasen, China). Protein signals were detected using an automatic chemiluminescence image analysis system and quantified with Image J.
Cell viability assessments
Cell viability assessments were conducted using the CCK-8 kit (Yeason, China). Vero E6 and Hela cells, treated as described, were subjected to the assay following the manufacturer’s guidelines. Post incubation at 37 °C, absorbance at 450 nm was measured using a Thermo Scientific Microplate Reader (Thermo, USA).
Protein purification
To express the full-length and transmembrane domain of SARS-CoV-2-E protein, a 6× his tag was added at the C-terminus. The protein, contained in the pET28a vector, was expressed in E. coli BL21 (DE3) pLysS competent cells (TransGen Biotech, China). The purification process was as previously described [18].
Planar lipid bilayers recording
Planar lipid bilayer recordings were performed as previously detailed. The bilayer comprised phosphatidylcholine (PC) and phosphatidylserine (PS) in a 3:2 ratio (Avanti, USA). Each side of the bilayer (trans and cis) received 1 mL of either a 500 mM KCl and 5 mM HEPES solution (pH 6.35) or a 50 mM KCl and 5 mM HEPES solution (pH 6.35). An electrochemical gradient and agitation facilitated protein insertion into the membrane. Currents were digitized using pClamp 10.2 software (Molecular Devices, CA, USA), disregarding opening times less than 0.5-1.5 ms. Single-channel conductance was determined through Gaussian function fitting.
Living cell imaging
Vero E6 cells were seeded at 3 × 103 per well in a 96-well cell culture microplate (Greiner, Germany). The subsequent day, the plasmid was transfected at 200 ng/well. Six hours post-transfection, the original medium was replaced with 5 µM Hoechst buffer to visualize living cells, with an additional 10 µM BE-33 added for one group. After 24 h of transfection, images were captured using a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek Instruments, Winooski, VT) at 20X objective in the DAPI, Texas Red, and Phase Contrast channels. Each group had over three wells, with six areas captured per well. Gen 5 software was used for image analysis to obtain cell count, red positive cell number (indicative of transfection), and red spot number (representing 2-E-EVs). After background removal, the Hoechst fluorescence threshold served as the primary mask (threshold > 5000, object size 10–50 µm), and Texas Red fluorescence as the secondary mask (threshold > 8000, size 10–50 µm). An expanded secondary mask identified red spots, considered 2-E-EVs, with diameters less than 1–8 µm and a fluorescence threshold > 8000.
Statistical analysis
All statistical analyses were done by GraphPad Prism 9.0 software and data was presented as mean ± SEM. Student's t-test was used to evaluate statistical significance. P value < 0.05 was considered statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.