The RNA pseudoknots in foot-and-mouth disease virus are dispensable for genome replication but essential for the production of infectious virus

Joseph C. Ward; Lidia Lasecka-Dykes; Chris Neil; Oluwapelumi Adeyemi; Sarah Gold; Niall McLean; Caroline Wright; Morgan R. Herod; David Kealy; Emma Warner; Donald P. King; Tobias J. Tuthill; David J. Rowlands; Nicola J. Stonehouse

To introduce mutations into the PK region, the pRep-ptGFP replicon plasmid was digested 121 with SpeI and KpnI and the resulting fragment inserted into a sub-cloning vector (pBluescript) 122 to create the pBluescript PK. PKs 3 and 4 were removed by digestion with HindIII and AatII 123 before insertion of a synthetic DNA sequence with PK 3 and 4 deleted. PKs 2, 3 and 4 were 124 deleted by PCR amplification using ΔPK 234 Forward primer and FMDV 1331-1311 reverse 125 primer, the resultant product was digested with HindIII and AatII and ligated into the 126 pBluescript PK vector. Complete PK deletion was achieved by introduction of an AflII site at 127 the 3′ end of the poly-C tract by PCR mutagenesis to create the sub-cloning vector, pBluescript 128 C11, which was then used to remove all the PKs by PCR mutagenesis using ΔPK 1234 forward 129 primer and FMDV 1331-1311 reverse primer. The modified PK sequences were removed from 130 the sub-cloning vectors and inserted into the pRep-ptGFP plasmid using NheI-HF and KpnI-131 HF.