Proposed structures of STNV-TED and of TED-like CITEs predicted in several viruses.

<p>Virus acronyms: <i>Satellite tobacco necrosis virus</i> (STNV), <i>Calibrachoa mottle virus</i> (CbMV), <i>Pelargonium chlorotic ring pattern virus</i> (PCRPV), <i>Elderberry latent virus</i> (ELV), <i>Pelargonium ring spot virus</i> (PelRSV), <i>Rosa rugosa leaf distortion virus</i> (RrLDV) and <i>Pelargonium line pattern virus</i> (PLPV). CbMV is a member of genus <i>Carmovirus</i> and PCRPV, ELV, PelRSV, RrLDV and PLPV are recommended to be included in the tentative new genus <i>Pelarspovirus</i>, all within family <i>Tombusviridae</i>. The region corresponding to the previously proposed STNV-TED as well as those corresponding to the previously proposed TED-like CITEs of CbMV, PCRPV and PLPV [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152593#pone.0152593.ref015" target="_blank">15</a>] are shown on a grey background.</p

Effect of mutations in the TED-CITE and/or in a hairpin within p27 ORF on translation of reporter-based genomic PLPV transcripts.

<p>(A) Putative secondary structure of the 5´-proximal 125 nt of PLPV according Mfold predictions. A potential long-range interaction between the apical loop of a hairpin (gHP3) predicted within p27 ORF and the apical loop of the 3´-CITE is shown. Nucleotides that can putatively pair are connected by dotted lines. (B) Translation efficiency of transcripts bearing mutations that disrupted or reconstituted the potential kissing-loop interaction between apical loops of gHP3 and the CITE. All mutants were generated from parental construct gFF and assayed in <i>N</i>. <i>benthamiana</i> protoplasts as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152593#pone.0152593.g005" target="_blank">Fig 5</a> legend. Only the sequences of the loops involved in the potential base-pairing are shown. The engineered nucleotide substitutions are on a gray background and putative G:U base pairs are in italics. (C) Translation efficiency of transcripts bearing mutations in the CITE outside of its apical loop. In B and C, levels of translation as a percentage of that of gFF construct are given. Results are from three experiments with standard deviations. Other details as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152593#pone.0152593.g005" target="_blank">Fig 5</a>.</p

Examination of the structure of the 3´-terminal region of PLPV through <i>in silico</i>, <i>in vitro</i> and comparative structural analyses.

<p>(A) Secondary structure of the 3´-terminal region of PLPV (bottom). The represented structure embraces the 3´-terminal sequence of the p37 gene and the entire 3´ UTR (arrow), contains several hairpins (HP1 to HP4) and corresponds to the optimal one according to Mfold predictions using the entire PLPV gRNA. Nucleotides likely involved in an interaction that presumably acts as repressor of minus-strand synthesis are within grey circles. Underlined nucleotides are predicted to base-pair with a segment of the gRNA out of the 3´-terminal region. HP4, that might be a CITE of the TED class, shows a SHAPE-derived flexibility profile (autoradiographs at the top) consistent with the Mfold predictions. In autoradiographs of SHAPE analysis, the lanes have been labelled as: N, for the NMIA treated RNA, D, for the DMSO treated RNA, G, C and A, for sequencing ladders. Residues in the putative TED and flanking region with high and medium reactivity to NMIA are denoted with red and green colors, respectively, whereas those with low or no reactivity to the reagent are in black. Bulges (B) and loops (L) of HP4 have been numbered on the secondary structure representation to facilitate comparison with SHAPE profile. The apical loop and a lateral bulge of the upper part of HP4 have been labelled as AL and BG, respectively. The region of HP4 previously proposed as putative TED-like CITE (15) is indicated by a square bracket with discontinuous line at the left side of the structure. (B) SHAPE profiles across a portion of the 3´-terminal region of the PLPV wild-type (wt) gRNA and a mutated (mut) gRNA carrying nucleotide replacements (in italics) in the apical loop of HP4. Strong NMIA reactivity of the apical loop in the mutant is observed when compared with the wt molecule. This result is in agreement with the involvement of the loop in RNA-RNA interaction as previously proposed [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152593#pone.0152593.ref015" target="_blank">15</a>]. The lanes have been labelled as in panel A and the sequencing ladders have been performed on the wt template. (C) Distribution of natural sequence heterogeneity along HP4. Twenty natural PLPV variants [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152593#pone.0152593.ref043" target="_blank">43</a>] were included in the analysis. Nucleotide replacements are in blue and the number of variants with a given mutation is shown in parentheses. Co-variations and mutations resulting in conversion of canonical Watson-Crick to wobble base pairs, or vice versa, in stems are boxed. Only two minority mutations affecting residue U3723 (marked by an asterisk), could lead to slight destabilization of a stem.</p

Schematic representation of the PLPV genome.

<p>PLPV gRNA contains five ORFs (represented by grey boxes) flanked by 5´- and 3´- UTRs (striped boxes). The viral RNA dependent-RNA polymerase, p87, is the ribosomal read-through (RT) product of p27, an auxiliary replication protein. PLPV sgRNA is tri-cistronic and serves as mRNA for expression of two small movement proteins, p7 and p9.7, and of protein p37, which functions as coat protein and as the suppressor of RNA silencing.</p

Assessment of infectivity of PLPV mutants.

<p>Uncapped transcripts corresponding to the gRNA of wt and CITE-related mutants of PLPV were inoculated onto <i>N</i>. <i>benthamiana</i> plants. Local leaves were collected at 15 days post-inoculation and viral accumulation was determined by Northern blot analysis. Bioassayed mutants are indicated above the lanes of the autoradiograph. A mock inoculated sample (lane M) was also included. Positions of PLPV gRNA and sgRNA are indicated at the right. Ethidium bromide staining of ribosomal RNAs is shown below the autoradiograph as loading control.</p

Assessment of the relevance of the 3´-TED and/or a 5´-hairpin, that can potentially establish a kissing-loop interaction, on reporter-based PLPV subgenomic translation.

<p>A) Putative secondary structure of the 5´-proximal 33 nt of PLPV sgRNA according Mfold predictions. The start codon of p7 ORF is indicated by an arrow. A potential long-range interaction between the apical loop of a 5´-hairpin (sgHP1) and the apical loop of the TED-like CITE is shown. Nucleotides that can putatively pair are connected by dotted lines. (B) <i>In vivo</i> translation assay of subgenomic PLPV transcripts. A parental subgenomic construct for assessment of translation efficiency was generated by replacing almost the entire p7 ORF by the Fluc reporter gene. This parental construct, named sgFF, is shown at the top and mutants derived from it are depicted below. One of the mutants had a shorter 5´-region (sgFF-s5R) and the others carried mutations that disrupted (sgFF-M1 and M3) or preserved (sgFF-M1/M3) the potential kissing-loop interaction between the 3´-CITE and the 5´-sgHP1. For the latter mutants, only the sequences of the loops involved in the potential base-pairing are shown. The engineered nucleotide substitutions are on a gray background and putative G:U base pairs in potential kissing-loop interaction(s) are in italics. Translation efficiencies were measured in protoplasts and the mean and standard error of the mean from three replicates is shown. Other details as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152593#pone.0152593.g005" target="_blank">Fig 5</a>.</p

Effect of the addition of a 5´-cap on translation of reporter-based genomic PLPV transcripts.

<p>Uncapped and capped transcripts from the wt construct gFF and from CITE mutants gFF-D2 and gFF-M2 (depicted in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152593#pone.0152593.g005" target="_blank">5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152593#pone.0152593.g006" target="_blank">6</a>, respectively) were generated and their translation efficiencies estimated through assay in <i>N</i>. <i>benthamiana</i> protoplasts. Translation efficiencies of transcripts are shown as percentages with respect to that of the uncapped gFF (set to 100%) The mean and standard error of the mean from three replicates are represented.</p

<i>In vitro</i> translation assay in WGE of wt and mutant genomic PLPV transcripts.

<p>A scheme of PLPV gRNA constructs carrying deletions (depicted as dashed lines) in the 3´-UTR is shown. Segments at the 3´-region forming potential hairpins (HP1-HP4) are indicated by small white rectangles on the wt construct. A representative autoradiograph showing p27 levels produced <i>in vitro</i> from each construct (top) and a graphic representation of such levels (bottom) are shown within an inset at the left. For the graphic representation, protein expression levels from wt gRNA were set to 100% and the translation efficiencies from other templates are indicated as percentages with respect the wt. Each percentage is shown as the mean and standard error of the mean from three replicates. Other details as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152593#pone.0152593.g001" target="_blank">Fig 1</a>.</p

STNV-TED and putative TED-like CITEs predicted in the 3´ UTR of several viruses (middle panel) can potentially base-pair with the 5´-region in the gRNA (upper panel) or sgRNA (lower panel) context.

<p>Virus acronyms as is <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152593#pone.0152593.g003" target="_blank">Fig 3</a>.</p

Bioassay of PLPV (wt and mutants) in <i>N</i>. <i>benthamiana</i> plants.

<p>Bioassay of PLPV (wt and mutants) in <i>N</i>. <i>benthamiana</i> plants.</p