Fig 1.
Self-folding of vRNA8 predicted by RNAstructure 5.3 using as constraints: strong reactivity of DMS; consensus base pairs from sequence and structure analysis (orange bars); SHAPE reactivities converted to pseudo- free energies.
Additionally there are marked results from microarray mapping in buffer A (300 mM NaCl, 5 mM MgCl2, 50 mM HEPES, pH 7.5) at 37°C and also from RNase H cleavage in the same buffer and temperature. Binding sites of probes are denoted by the middle nucleotide of the five nucleotides complementary in the RNA. Possible regions of tertiary interactions are marked by letters in open circles—the same letter marks one interaction (see text). Regions with no read-out by chemical mapping are: 807–875 (NMIA) and 835–875 (DMS). The numbering of vRNA8 is from its 5’ end. The template for the AUG start codon is nucleotides 849–847.
Fig 2.
Conservation of vRNA8 self -folding in type A viruses.
Colors indicate percentage of canonical base pairing preserved across vRNA segment 8 of type A strains. Compensating changes occur for base pairs U60/G77, C63/G74, C99/G117, C661/G814, C664/G811, U667/A808, C694/G782, A697/U779, and A703/U773. The numbering of vRNA8 is from its 5' end.
Fig 3.
RNAstructure5.3 predicted probability of nucleotides being paired (colored lines) or single stranded (colored circles) in vRNA8 self-folding.
Probability lower than 50% is not colored. The partition function calculation incorporated restraints from strong reactivity of DMS, consensus basepairs from sequence and structure analysis and SHAPE reactivities converted to pseudo-energy.
Fig 4.
Self-folding of vRNA8 marked with regions not accesible for probes on microarray (buffer A) (see Table F in S1 File).
RNase H cleavage sites for selected DNA oligonucleotides are also marked.
Table 1.
RNase H confirmed strong binding sites in vRNA8 for microarray probes.
Table 2.
Deduced medium binding sites in vRNA8 for microarray probes.
Fig 5.
Self-folding of mini-vRNA8 predicted by RNAstructure 5.3 using as constraints: strong reactivity of DMS and SHAPE reactivities converted to pseudo-energy.
Additionally there are marked results from microarray mapping in buffer A (300 mM NaCl, 5 mM MgCl2, 50 mM HEPES, pH 7.5) at 37°C. All symbols are the same as in Figs 1 and 4. The regions without readout of chemical mapping results are: 327–376 (826–875) (NMIA), and 322–376 (821–875) (DMS). Numbering of mini-vRNA8 is from its 5’ end and numbers in parenthesis correspond to respective nucleotides in vRNA8. Nucleotides 183–187, 5’GGAUC, were introduced for cloning (see Materials and methods). Nucleotides 1–182 and 188–376 correspond to wild type. Efficient packaging of a segment 8 encoding only GFP protein required nucleotides 1–177 and 198–376 (mini-vRNA8 nomenclature) [45].
Fig 6.
Aligned results of hybridization of vRNA8 (blue) and mini-vRNA8 (orange) to isoenergetic microarrays.
All complementary sites for binding probes are shown and grouped in regions of binding.
Table 3.
Possible tertiary interactions in vRNA8 structure.