Furin

The Furin reactivation assay was performed as previously described^{34 (link)}. 2x10⁶ cells were transduced with lentivirus (as described above) and, 48 h later, were infected with DV-2 (16681 strain) for 48 h followed by collection of the serum (total volume 10 mL for each sample). Virus particles in the serum were purified by centrifugation through 2 mL 20% sucrose containing NTE buffer (120 mM NaCl, 12 mM Tris-HCL pH 8, 1 mM EDTA) for 4 h at 10,000xg at 4°C. Supernatant was then removed and the resulting pellet was resuspended in 50 μl NTE buffer. Purified DV particles in NTE buffer were mixed with 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) at a 1:1 ratio to adjust the pH to 6.0. Furin (New England Biolabs Inc.) was added at a final concentration of 0.4 U/μL, and the mixture was incubated at 30°C for 16 h in the presence of 3 mM CaCl₂. The samples were then mixed with neutralization buffer (120 mM NaCl, 100 mM Tris at pH 8.0) at a 1:1 ratio. Virus particle infectivity was determined by PFU assay as described above.

N-glycans were removed by endo-β-N-acetylglucosaminidase H (Endo H, New England BioLabs, Ipswich, MA) or peptide-N-glycosidase F (PNGase F, Sigma, St. Louis, MO). Propeptide were cleaved by furin (New England BioLabs). For Endo H digestion, aliquots of total cell lysates from vehicle- or TG-treated cells were boiled for 5 min in 0.5% SDS and 40 μM DTT. After cool down to room temperature, denatured proteins were digested for 4 h at 37 °C with or without Endo H (20 units/μg protein) in buffer containing 50 mM sodium citrate (pH 5.5), 0.5 mM PMSF, and proteinase cocktail. For PNGase F digestion, aliquots of the same set of protein samples were boiled for 5 min in protein denaturing buffer (50 mM phosphate buffer, pH 7.5, 0.1% SDS, and 0.05 mM β-mercaptoethanol) and cooled down to room temperature. Denatured proteins were digested for 4 h at 37 °C with or without PNGase F (0.2 units/μg protein) in the presence of 0.75% Trion X-100. For furin digestion, aliquots of Triton X-100 soluble proteins from vehicle- or TG-treated cells were digested for 18 h at 30 °C with or without furin (0.04 units/μg protein) in buffer containing 1 mM CaCl₂, and 1 mM β-mercaptoethanol). Following digestions, reactions were stopped by adding Laemmli buffer and subjected to Western Blot analysis.