A conserved predicted pseudoknot in the NS2A-encoding sequence of West Nile and Japanese encephalitis flaviviruses suggests NS1' may derive from ribosomal frameshifting

Abstract

Japanese encephalitis, West Nile, Usutu and Murray Valley encephalitis viruses form a tight subgroup within the larger Flavivirus genus. These viruses utilize a single-polyprotein expression strategy, resulting in ~10 mature proteins. Plotting the conservation at synonymous sites along the polyprotein coding sequence reveals strong conservation peaks at the very 5' end of the coding sequence, and also at the 5' end of the sequence encoding the NS2A protein. Such peaks are generally indicative of functionally important non-coding sequence elements. The second peak corresponds to a predicted stable pseudoknot structure whose biological importance is supported by compensatory mutations that preserve the structure. The pseudoknot is preceded by a conserved slippery heptanucleotide (Y CCU UUU), thus forming a classical stimulatory motif for -1 ribosomal frameshifting. We hypothesize, therefore, that the functional importance of the pseudoknot is to stimulate a portion of ribosomes to shift -1 nt into a short (45 codon), conserved, overlapping open reading frame, termed foo. Since cleavage at the NS1-NS2A boundary is known to require synthesis of NS2A in cis, the resulting transframe fusion protein is predicted to be NS1-NS2A(N-term)-FOO. We hypothesize that this may explain the origin of the previously identified NS1 'extension' protein in JEV-group flaviviruses, known as NS1'.

Figure 1

Conservation at synonymous sites in Japanese encephalitis and related viruses. Conservation at synonymous sites was calculated for an input alignment comprising the polyprotein CDSs from the seven JEV-group sequences listed in the caption to Figure 2. (A) Panels 1–3 show the positions of stop codons (blue triangles) in the three forward reading frames. The +0 frame is the polyprotein frame and is therefore devoid of stop codons. Alignment gaps are indicated in green. Panel 4 shows the difference between the expected number (assuming neutral evolution) and observed number of base substitutions at synonymous sites, summed over a phylogenetic tree, and averaged over a 25-codon sliding window. Panel 5 shows the estimated standard deviation for the statistic in panel 4 – major dips tend to correspond to alignment gaps (fewer pairwise sequence comparisons to sum over), while the rise at each end of the alignment corresponds to the shorter terminal windows over which statistics are averaged. Panel 6 is an approximation of the p-value corresponding to the statistic in panel 4, albeit subject to the assumption of normal distribution. Panel 7 shows a map of the JEV genome, indicating the position of the putative -1 ribosomal frameshift site, and the putative transframe protein which may equate to NS1'. (B) Zoom-in of the region corresponding to the conservation peak in the NS2A CDS with a 15-codon sliding window. Note the highest level of conservation corresponds precisely to the region covered by the predicted pseudoknot.

Figure 2

Putative stimulatory elements for ribosomal -1 frameshifting. (A) The slippery heptanucleotide and predicted 3' RNA pseudoknot structure for JEV [GenBank:NC_001437]. (B) Sequence alignment of different JEV-group sequences, showing the conserved presence of a slippery heptanucleotide (Y CCU UUU; orange) and potential to form a 3' RNA pseudoknot. Base-pairings in stem 1 are indicated with '()'s and yellow background while base-pairings in stem 2 are indicated with '[]'s and green background. Base substitutions that preserve base-pairings or the slippery heptanucleotide are highlighted in red. Viruses: Japanese encephalitis (JEV) – [GenBank:NC_001437]; West Nile (WNV; lineage I) – [GenBank:NC_009942]; West Nile (WNV; lineage II) – [GenBank:NC_001563]; Kunjin – [GenBank:AY274504]; Murray Valley encephalitis (MVEV) – [GenBank:NC_000943]; Alfuy – [GenBank:AY898809]; Usutu – [GenBank:NC_006551].

Figure 3

Peptide sequences for the putative transframe extension of NS1. Predicted peptide sequences for NS2A^N-FOO – the putative transframe extension of NS1 – for various JEV-group viruses. The shift-site amino acids are indicated in the orange rectangle, and the foo termination codon is represented by an '*'. See caption to Figure 2 for virus names.