Ultracentrifugation

To generate HCV pseudo-particles, 293T cells were transfected with expression vectors encoding the viral components (see Fig. 1 B), i.e., E1E2 glycoproteins, retroviral core proteins, and packaging-competent GFP- or nlslacZ-containing retroviral transfer vectors. In brief, the Gag-Pol packaging construct (8.1 µg), the transfer vector construct (8.1 µg), and the glycoprotein-expressing construct (2.7 µg) DNAs were transfected into 2.5 × 10⁶ 293T cells seeded the day before in 10-cm plates using a calcium phosphate transfection protocol (CLONTECH Laboratories, Inc.), as described previously (11 (link)). The medium (8 ml/plate) was replaced 16 h after transfection. Supernatants containing the pseudo-particles were harvested 24 h later, filtered through 0.45-µm pore-sized membranes, and used in infection assays. Purified virus samples were obtained by ultracentrifugation of 10-ml viral supernatants through a 1.5-ml 20% sucrose cushion in an SW 41 Beckman rotor (25,000 rpm, 2.5 h, 4°C). Viral pellets were suspended in 50 µl PBS. Immunoblots of producer cell lysates and purified pseudo-particles were performed as described previously (15 (link)). Fractionation of the sucrose cushion purified viral pellets was achieved by an overnight equilibrium density centrifugation in a 20–60% sucrose gradient at 35,000 rpm and 4°C in a Beckman SW 41 rotor. Fractions of 0.7 ml were collected, precipitated with TCA, and analyzed by Western blotting.

293FT cells were cultured in DMEM (#61965026; Thermo Fisher Scientific) with 5% PS and 10% FBS. 293FT cells were plated at 0.8 million cells in a 6-well plate and transfected with 3 μg of DNA complexed in 8 μl of TransIT-293 (#MIR2706; Mirus Bio) per well. The ratio of DNA used for transfections was as follows: 0.2 μg HXB2 env, 0.2 μg CMV-VSVG, 1 μg psPAX2, and 1.6 μg of pTRIP-SFFV or pLKO.1 lentivector. The ratio of plasmids for the production of HIV-1 and HIV-2 BFP single-round reporter viruses was 0.2 μg HXB2 env, 0.2 μg CMV-VSVG, and 2.6 μg HIV plasmid. SIV-VLPs were produced using 0.4 μg CMV-VSVG with 2.6 μg pSIV3⁺. Lentiviruses for MDMs were prepared by plating 7 million 293FT cells in T75 flasks and transfected with 8.35 μg DNA complexed in 116 µl PEImax (0.75 mM; #24765; Polyscience) per flask. The ratio of DNA used for transfection includes 3 μg psPAX2, 1.25 μg CMV-VSVG, and 4.10 µg pTRIP-SFFV-GFP. For SIV-VLP production for MDMs, 2.5 μg CMV-VSVG with 8.2 μg pSIV3⁺ was used. 18 h following transfection, media was removed and replenished with fresh media (3 ml for T cells and MDDCs or 8.5 ml for MDMs). 24–26 h later, viral supernatants were harvested, filtered using 0.45 μM filters, and used fresh or stored at −80°C.
For CAR expression, rLV.EFA.19BBz CAR lentivirus was produced using pLV, pHIV-GagPol, and pEnv plasmids and concentrated by ultracentrifugation (Flash Therapeutics). Titer was determined by serial dilution on activated human T cells.