Coronavirus nucleocapsid proteins assemble constitutively in high molecular oligomers

Cong, Yingying; Kriegenburg, Franziska; de Haan, Cornelis A. M.; Reggiori, Fulvio

Scientific RepoRts | 7: 5740 | DOI:10.1038/s41598-017-06062-w Bacterial extracts. Transformed Escherichia coli BL-21 were grown in 125 ml of LB medium (0.5% yeast extract, 1% tryptone, 1% NaCl) to late exponential phase and after inducing protein expression by addition of 0.5 mM isopropyl-β-D-thiogalactopyranoside, cells were grown at 37 °C or 20 °C for 4 h or 16 h, respectively. Bacteria were harvested, resuspended in 4 ml lysis buffer (PBS, 5 mM DTT, 1 mg/ml lysozyme, 1 mM PMSF, 10% glycerol, 1% Triton X-100 and complete protease inhibitor (Roche) and lysed by two sonication rounds of 10 sec using a Branson sonicator (Danbury, Connecticut, United States). The bacterial lysates were cleared by centrifugation at 15,000 × g for 10 min at 4 °C and passed through a 0.45 µm filter. For purification of GST fusion proteins, lysates were incubated with 125 µl of glutathione (GSH) Sepharose (4B, GE Healthcare), which had been pre-washed in PBS. Where indicated, lysates were incubated with 40 mg/ml RNase A (Invitrogen, Carlsbad, CA) for 30 min on ice prior to addition to the GSH Sepharose. Cell extracts from bacteria expressing the 6xHis-tagged proteins were used either directly for pull-down experiments or for the purification of the fusion proteins with nickel Sepharose (6 Fast Flow, GE Healthcare) after incubation in presence or absence of 40 mg/ml RNase A for 30 min on ice. Complete hydrolysis of RNA was verified by sample analysis on an agarose gel followed by nucleic acid staining with Midori Green (GC Biotech, Netherlands) or determination of the A260/A280 ratio through measurement of A260 and A280 using a NanoDrop Spectrophotometer (Implen, Germany) 54-56 . Cell extracts. For the preparation of cell extracts, LR7 cells grown on 10 cm dishes were mocked-treated or inoculated with MHV at a MOI of 1 and after 8 h, they were lysed by 5 min sonication in 1.2 ml of PBS buffer supplemented as described above. Supernatants were then cleared by centrifugation at 15,000 × g for 10 min at 4 °C and passed through a 0.45 µm filter. RNase A treatments were carried out by incubating 200 µl of cell extract with 40 mg/ml of enzyme for 30 min on ice, immediately prior to pull-downs.