Sw40ti

Rate zonal centrifugation was performed on 12 ml linear 10–35% sucrose gradients in PBS at pH 7.4, as previously described^{41 (link)}. 500 μl samples were layered on top of the gradient and centrifuged at 21000 g for 90 minutes at 15 °C in a Beckman L-90 ultracentrifuge, using a Beckman SW40 Ti swing-out rotor. After centrifugation, tubes were fractionated from the top of the gradient and 24 fractions collected. Pelleted material at the bottom of the tube was recovered through solubilization in 6 M urea (VWR, Leicestershire, UK). The sucrose concentration of each fraction was determined through measurement of refractive index. Fractions were then analyzed for mucin distribution via immunodetection.

EVs obtained from HMC-1 cells were labeled with the PKH67 Green Fluorescent Cell Linker Kit (Sigma Aldrich) as per the manufacturer’s protocol. The labeled EVs were loaded onto the bottom of an iodixanol density gradient (0, 20, 30, and 50% iodixanol) and centrifuged at 28,000 rpm for 2 h in a swinging bucket rotor (SW40Ti, Beckman Coulter). The EVs floating over the interphase (20–30%) were collected and washed in PBS followed by centrifugation at 120,000×g for 3 h (Type 45 Ti rotor, Beckman Coulter). A549 cells were grown on coverslips at 15,000 cells/cm² for 24 h. The labeled EVs were incubated with the A549 cells grown on the coverslip for 2 h or for 16 h. The cell membranes and nuclei were stained with the Image-IT LIVE kit (Invitrogen, Thermo Fisher Scientific) using Alexa Fluor-594 wheat germ agglutinin and Hoechst 33342, respectively, according to the manufacturer’s protocol. The cells were fixed in a paraformaldehyde (3.5%) solution for 10 min and washed before the cover slip containing the cells was mounted on a slide and imaged under a structural illumination microscope (Zeiss Elyra 3D SIM, Germany).

Prior to cell harvesting, 100 µg/ml cycloheximide was added and incubation continued for another minute to stall ribosomes on the mRNA. After removal of supernatant, pellets were resuspended in 5 ml breaking buffer (20 mM Tris-HCl pH 7.5, 100 mM KCl, 0.5% NP40, 2 mM MgCl₂, 200 µg/ml heparin, 100 µg/ml cycloheximide, 2 mM DTT, 0.5 mM PMSF) on ice and transferred to corex tubes. Pellets were then resuspended in 1 ml breaking buffer and 4 µl RNaseOut and approximately 500 µl worth of glass beads added. Cells were lysed by vortexing corex tubes 10 times for 1 min with 1 min on ice in between. Supernatant was then transferred to 2 ml tubes and stored at −80°C until ready for further use.
Linear sucrose gradients were prepared in Beckman Coulter 14 × 96 mm centrifuge tubes with sequential loading of sucrose stocks in gradient buffer (50 mM Tris-HCl pH 7.0, 50 mM NH₄Cl, 0.5% NP40, 4 mM MgCl₂, 1 mM DTT and 100 µg/ml cycloheximide). From top to bottom, tubes contained 2.5 ml 17.5%, 2.5 ml 25%, 2 ml 33%, 2 ml 41% and 2.5 ml 50% sucrose solutions. Lysate was loaded to the top of the sucrose gradients and spun in a swinging bucket rotor Beckman Coulter SW40TI in a L-90K Preparative Ultracentrifuge at 38000 rpm for 2.5 hr at 4°C, max acceleration, no break. Gradients were fractionated by upward displacement with 60% sucrose at 3 ml/min and absorbance was monitored at 254 nm.

HSV-1 nuclear capsids were prepared from 40 × 175 cm² flasks with BHK-21 cells infected with 0.01 PFU/cell (3–4 x 10⁴ PFU/mL) for about 2.5 days (Anderson et al., 2014 (link); Radtke et al., 2010 (link); Radtke et al., 2014 (link); Snijder et al., 2017 (link); Wolfstein et al., 2006 (link)). VZV nuclear capsids were harvested from infected MeWo cells cultured in 5–10 x 175 cm² flasks at maximum syncytia formation but before cell lysis. The cells were harvested, resuspended in MKT buffer (20 mM MES, 30 mM Tris, pH 7.4, 100 mM KCl), snap-frozen, and stored at –80 °C. Nuclear A, B, and C capsids were separated by sedimentation at 50,000 x g and 4 °C for 80 min (SW40Ti, Beckman Coulter) on linear 20% to 50% sucrose gradients in TKE buffer 20 mM Tris, pH 7.5, 500 mM KCl, 1 mM EDTA; diluted in three volumes of TKE supplemented with 2 mM DTT and PIs (Roche cOmplete). The capsids were sedimented in BSA-coated centrifuge tubes at 110,000 g at 4 °C for 20 min (TLA-120.2), resuspended in BRB80 buffer supplemented with 100 mg/mL RNase (Roth, Germany), 0.1 U/mL DNase I (M6101, Promega, USA), 10 mM DTT, and PIs, sedimented again, and resuspended in CBB with PIs.

iSLK cells latently infected with mini-TurboID-KSHVs were seeded in 8 to 10 15-cm dishes, stimulated with 1 µg/ml of doxycycline and 3 mM sodium butyrate for 24 h, and further incubated with culture media without stimuli for 72 h. The culture supernatant was centrifuged using a Beckman SW28 rotor (25,000 rpm for 2 h) with a 25% sucrose cushion. Virus pellets were dissolved in DMEM and further purified by discontinuous sucrose gradient (25 to 60%) centrifugation using a Beckman SW40Ti rotor (21,000 rpm for 16 h). Virus pellets were dissolved in DMEM for infection studies. HEK293FT cells were infected with 1 genomic copy of virus per cell in DMEM. Twenty-four hours postinfection, cells were washed with PBS and incubated for 24 h in complete media. After 48 h postinfection, cells were trypsinized and washed twice with PBS. Cells were resuspended in PBS containing 1% bovine serum albumin (BSA) and 1 mM EDTA, and recombinant viral infection was analyzed with flow cytometry (BD Accuri) and FlowJo software.

The sucrose gradient was prepared with solutions containing 10, 20, 30, 40, and 50 % (w/v) sucrose in 50 mM Tris-HCl at pH 7.4. 2.5 ml of each solution were used to form the gradient in a 14 ml Ultra-Clear™ centrifuge tube (Beckman Coulter, Brea, CA, USA), with the highest sucrose concentration at the bottom of the tube. The cell-free extract was layered on top and the gradients were centrifuged at 85,365 x g (average relative centrifugal force in a Beckman Coulter SW 40 Ti swing bucket rotor) for 16 h at 4 °C [81 ]. The resulting gradient fractions were collected in the same pattern for every individual gradient.
For proteomic analysis, the buffer was exchanged to 50 mM Tris-HCl (pH 7.9) without sucrose using 10 kDa mass cutoff centrifugal filters (Sartorius Vivaspin 500, Sartorius AG, Göttingen, Germany), the samples were then brought to comparable protein concentrations and submitted to the Proteomics Facility of the University of Konstanz for protein mass fingerprinting. For electron microscopy, 5 mM Tris -HCl (pH 7.9) without sucrose was used.