Bacterial artificial chromosome (BAC) pBeloBAC11 (62 (link)), provided by H. Shizuya, California Institute of Technology, Pasadena, CA, was used to assemble recombinant MERS-CoV and MERS-CoV mouse-adapted (MERS-MA30) infectious complementary DNA (cDNA) clones. This plasmid is a low–copy number plasmid (one, maximum two copies per cell) based on the Escherichia coli F factor (63 (link)) that allows the stable maintenance of large DNA fragments in bacteria. E. coli DH10B (Gibco/BRL) cells were transformed by electroporation using a MicroPulser unit (Bio-Rad) according to the manufacturer’s instructions. BAC plasmid and recombinant BACs were isolated and purified using a large-construct kit (Qiagen), following the manufacturer’s specifications.
Two plasmids were used for the expression of the MERS-CoV E protein: pcDNA3.1 for constitutive expression (Invitrogen; Thermo Fisher Scientific) (64 (link)) and TRE-Auto-rtTA-V10-2T for inducible expression provided by A. Das and B. Berkhout, Academisch Medisch Centrum Universiteit van Amsterdam, Amsterdam, The Netherlands (65 (link)).
Two plasmids were used for the expression of the MERS-CoV E protein: pcDNA3.1 for constitutive expression (Invitrogen; Thermo Fisher Scientific) (64 (link)) and TRE-Auto-rtTA-V10-2T for inducible expression provided by A. Das and B. Berkhout, Academisch Medisch Centrum Universiteit van Amsterdam, Amsterdam, The Netherlands (65 (link)).
