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

The FMDV 5′ UTR is the largest known picornavirus UTR, comprising approximately 1300 71 nucleotides and containing several highly structured regions. The first 360 nucleotides at the 5′ 72 end are predicted to fold into a single large stem loop termed the S-fragment, followed by a The PKs were originally predicted in 1987 and consist of two to four tandem repeats of a ~48 86 nucleotide region containing a small stem loop and downstream interaction site (figure 1B) 87 (12). Due to the sequence similarity between the PKs (figure 1C), it is speculated that they 88 were formed by duplication events during viral replication, probably involving recombination. 89 Between two and four PKs are present in different virus isolates but no strain has been 90 identified with less than two PKs, emphasising their potential importance in the viral life cycle 91 (19, 20) . The presence of PKs has been reported in the 5′ UTR of other picornaviruses such as 92 author/funder. All rights reserved. No reuse allowed without permission. can occur in the absence of PKs at least one is required for wild-type (wt) replication. 104 Furthermore, competition experiments showed that extra copies of PKs conferred a replicative 105 advantage to genomes. Although replicons and full-length genomes lacking PKs were 106 replication-competent, no infectious virus was rescued from genomes containing less than one 107 PK copy. This is consistent with our earlier report describing the presence of putative 108 packaging signals in the PK region (22). 109 110 author/funder. All rights reserved. No reuse allowed without permission. Plasmid construction. 117 The FMDV replicon plasmids, pRep-ptGFP, and the replication-defective polymerase mutant 118 control, 3D-GNN, have already been described (10).