Text S1 - Rapid Evolution of Virus Sequences in Intrinsically Disordered Protein Regions

Derivation of cDNA for Nodamura virus RNA1 (protocol). Fig. S1. In vivo assay to test the influence of various 2A peptide fusions to the polymerase C-terminus. A. Construct Design. Mutant polymerase-encoding replicon NodaF2A-Luc/GAA was co-transfected with a plasmid which supplied a polymerase in trans from the EF1a promoter. The polymerase-expressing plasmid had all non-coding RNA1 sequences removed and the last third of the RDRP ORF coded with synonymous codons; the synonymous region is depicted in grey stripes. Each transfection also contained a renilla luciferase-expressing plasmid as a normalization control (not shown). Firefly luciferase counts from the pEF6-PolWT transfection are taken as 100%, while polymerases bearing various 2A versions are shown as % of wt value. B. Co-translational “cleavage” efficiency of “T2A” and “F2A” peptides in replicons (NodaT2A-GFP, NodaT2A-GFP/GAA) and expression plasmids (pEF6-PolT2A-GFP and pEF6-PolF2A-GFP). The Western blot was probed for GFP. pEF6-GFP is used as a positive control; (-) denotes a lane with lysate from untransfected cells. Arrow indicates the unprocessed PolF2A-GFP polypeptide. Fig. S2. Sequence of the pNoda-B2GFP replicon in the B1/B2 region. Wildtype sequence of RNA1 is maintained 5′ of the AscI site (in grey); accordingly, B2 ORF is outlined in purple and A/B1 ORF is in yellow. Insertion of GFP (green, in frame with B2) leads to the replacement of the wildtype ORF A 3′ end with the de novo produced FPG ORF (red). Table S1. Differences between Mag115 strain sequence and the reference (passaged) sequence. Table S2: Links to viral sequences used in this study. Table S3: Relative composition (% of each amino acid) in disordered regions. (DOCX)