Using “en passant recombineering” (recombineering by
mutagenesis) (54 (link)), 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 (link)). 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 × 106BHK-Tet-SARS-N cells, which express the SARS-CoV N protein after
>4 h of induction with 2 μm doxycycline (53 (link)). 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.
mutagenesis) (54 (link)), 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 (link)). 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 × 106BHK-Tet-SARS-N cells, which express the SARS-CoV N protein after
>4 h of induction with 2 μ
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.
