Figure 1.
Conserved shunt configurations in the RTBV and RTSV leader sequences.
The primary and secondary structures of RTBV (A) and RTSV (B) leaders preceding the first large viral ORF (ORF I) are shown schematically. Short ORFs within the leaders are indicated by boxes, with internal AUGs indicated by vertical lines. Arrows under the leader line define the ascending and descending arms that form the base section of the large stem-loop structure. The stem-loop structures are predicted by the MFold program (Wisconsin GCG package) at 25°C and schematically drawn below the leader primary structures. The 5′- and 3′-sequences flanking the main structure are shown in open conformation. The stable structural element at the stem base (stem basal helix) and adjacent regions, are enlarged and their sequences shown. The nucleotide numbering is from the RNA 5′-end. The 5′-proximal short ORF (sORF1) is boxed. The sORF1 AUG and the non-AUG start codons in the shunt landing site are underlined. The identical nucleotide stretches/motifs in the shunt take-off and landing sites are highlighted in bold. Nucleotide substitutions that occur in five isolates of RTSV are indicated with arrows.
Figure 2.
Translation downstream of the RTSV and RTBV leaders is regulated by the first sORF.
Relative values of CAT expression downstream of the wild type and mutated versions (‘KO start’ and ‘KO stop”) of the RTSV (top panel) and RTBV (bottom panel) leaders in the two translation systems are given. Expression from the wild type RTSV construct in O. sativa (rice) protoplasts and in the wheat germ (WG) in vitro system is set to 100%. The sORF1 region of the leaders in each construct is shown schematically; point mutations are indicated with crosses and sORFs with boxes.
Figure 3.
Translation downstream of the RTSV leader is initiated by shunting but not internal initiation.
Relative values of CAT expression downstream of the wild type (“Wild type”) and sORF1-mutated versions (“KO start” and “KO stop”) of the RTSV leader carrying the KS at the 5′ end or in the middle region in O. sativa (rice) protoplasts are given. CAT expression from the wild-type leader construct in the absence of KS is set to 100%. For each construct, the RTSV leader preceding the polyprotein ORF (ORF I) fused to the CAT reporter ORF is depicted as thick line: the sORFs are indicated by boxes, point mutations shown with crosses, KS insertions indicated with thick vertical lines.
Figure 4.
Integrity of the stem base secondary structure is essential for RTSV shunting.
Twelve point mutations in either ascending (Disrupt L) or descending (Disrupt R) arm of the RTSV stem base secondary structure are shown on the left side. A combination of these mutations (Restore L+R) restores stable secondary structure. On the right side, relative values of CAT expression downstream of the wild type (“Wild type”) and the stem base-mutated versions (“Stem Disrupt L”, “Stem Disrupt R” and “Stem Restore L+R”) of the RTSV leader [or its variant with the Kozak stem (KS) sequence in the middle part] in O. sativa (rice) protoplasts are given. CAT expression from the wild-type leader construct is set to 100%.