Optiprep

To generate VSG-containing conditioned medium (CM), washed cells were resuspended at 10⁷/ml in HMI-9 medium and incubated for 6 h at 37°C. Following incubation, cells were removed on Costar SpinX centrifuge filters (0.45 μm; Corning Inc., Salt Lake City, UT), and the cleared CM was supplemented with protease inhibitor cocktail (PIC; 2 μg/ml of leupeptin, antipain, chymostatin, pepstatin A, and aprotinin). For analytical step floatation gradients, CM was adjusted to 40% OptiPrep (Sigma-Aldrich, St. Louis, MO), and 350 μl was overlaid with 35% OptiPrep phosphate-buffered saline (PBS) (600 μl) and 10% OptiPrep-PBS (450 μl) in thick wall polycarbonate TLS-55 tubes (Beckman Coulter Inc., Brea, CA). After centrifugation (TLS-55 rotor, 200,000 × g, 2 h, 4°C), gradients were manually fractionated from the top (5 × 280-μl fractions). Samples were fractionated by SDS-PAGE and immunoblotted with anti-VSG221 VSG or anti-CRD antibodies (12 (link)).

NEC sensitivity to membrane curvature was tested using coflotation as described previously (120 (link)). Briefly, 1.5 μg NEC was incubated with or without large unilamellar vesicles (LUVs) (POPC, POPS, and POPA mixed in a 3:1:1 molar ratio as previously described [21 (link)]) at room temperature for 20 min in 50 ml phosphate-buffered saline (PBS). KCl was added to 200 mM concentration to reduce nonspecific protein-membrane interactions, and samples were incubated for 15 min at room temperature. OptiPrep (Sigma) was added to a final concentration of 30% in a 500-ml volume. Samples were placed at the bottom of a 5-ml centrifugation tube (Beckmann) and overlaid with 4 ml 15% OptiPrep and 500 ml 3% OptiPrep in PBS. The samples were next centrifuged in a Beckman SW-55 Ti rotor at 246,000 × g for 3 h at 4°C, and 1-ml fractions were collected beginning at the top. Protein was precipitated with 20% trichloroacetic acid for 30 min on ice. Sample was washed with 750 μl cold acetone and then spun in a tabletop centrifuge for 10 min at 14,000 rpm. This was repeated for a total of 3 washes. Samples were then analyzed by Western blotting for UL31 as previously described (21 (link)). The standard error of the mean is reported from at least two individual experiments. Data were plotted using GraphPad Prism 9.0.

For initial experiments, 40-nmol total lipid was incubated with 200 pmol FisB ECD for 1 hour at room temperature in a total volume of 100 μl. A total of 200 μl of 60% Optiprep (iodixanol, Sigma-Aldrich) was added to the sample, creating a 40% Optiprep solution. The sample was then layered at the bottom of a 5 mm × 41 mm Beckman ultracentrifuge tube (#344090) and overlaid with 200 μl of 20% Optiprep and finally 150 μl of buffer (Fig 4C). Liposome-bound proteins co-float to a light density, while unbound proteins pellet upon ultracentrifugation for 1.5 hours at 48 krpm. Fractions were collected as shown in Fig 4C, and the amount of recovered protein was determined by SDS-PAGE (Nu-PAPGE 12% Bis-Tris gel, Thermo Fisher Scientific) stained with SYPRO Orange (Invitrogen).

SV40 was purified using the OptiPrep (60% stock solution of iodixanol in water; Sigma) gradient centrifugation method described previously in [39 (link)]. Briefly, viral genome transfected CV-1 cells were lysed in a buffer containing 50 mM Hepes pH 7.5, 150 mM NaCl and 0.5% Brij 58 for 30 min on ice, and the supernatant was collected after centrifugation at 20,000x g for 10 min. The supernatant was placed on top of a discontinuous OptiPrep gradient of 20% and 40%, and centrifuged at 49,500 rpm for 2 h at 4°C in an SW55Ti rotor (Beckman Coulter, Indianapolis, IN). A white interface formed between 20% and 40% OptiPrep was collected, and aliquots were stored at -80°C for future use. Purified BKV and antibody against BKV large TAg (pAB416) were generous gifts from Dr. Michael Imperiale (University of Michigan).

Wild-type coxsackievirus A21, Kuykendall strain, was provided by Viralytics Ltd. (Sydney, Australia) or propagated in-house from wild-type CVA21 obtained from ATCC® (ATCC®VR-850™). For propagation, supernatants were harvested following CVA21 infection of Mel624 cells for 24 h. CVA21 was pelleted by centrifugation at 36000 rpm for 2 h (SW45 rotor, Optima™ L-80 ultra-centrifuge, Beckman Coulter) and harvested virus was purified using OptiPrep™ density gradient centrifugation, 35–15% gradient (36,000 rpm, 1.5 h, SW41 Ti rotor). Viral titer was determined using a standard plaque assay on Mel624 cells.