SARS-CoV Reverse Genetics
Using “en passant recombineering” (recombineering by mutagenesis) (54), mutations in the nsp10-, nsp14-, and nsp16-coding regions of SARS-CoV isolate Frankfurt-1 were engineered in prSCV, a pBeloBac11 derivative containing a full-length cDNA copy of the viral genome (55). The DNA of such BAC clones was linearized with NotI, extracted with phenol-chloroform, and transcribed with the mMessage-mMachine® T7 (Ambion) using 2 µg of DNA template in a 20-µl reaction. Full-length viral RNA was precipitated with LiCl according to the manufacturer's protocol, and 6 µg was electroporated into 5 × 106 BHK-Tet-SARS-N cells, which express the SARS-CoV N protein after> 4 h of induction with 2 µm doxycycline (53). Electroporation was done using the Amaxa Nucleofector (Lonza), Nucleofector Kit T, and program T-020 according to the manufacturer's instructions. Cells were mixed in a 1:1 ratio with Vero-E6 cells and seeded on coverslips for immunofluorescence microscopy and for analysis of virus production. Each mutant was launched twice from independently generated BAC clones. All work with live SARS-CoV was performed inside biosafety cabinets in a biosafety level 3 facility at Leiden University Medical Center.