PMC

Potential downstream RNA secondary structures in all sequences analysed. (Continued from Figure 3.)

Alphavirus genome map. The position of the -1 ribosomal frameshift site is indicated. Nucleotide coordinates are for SFV ([GenBank:NC_003215]; 11442 nt).

Immunoprecipitation of SFV 6K and/or TF proteins for WT and TF^- viruses. SFV-infected cells were labelled with [³⁵S]Met/Cys and cell lysates (1 hr and overnight, o/n) and purified virions were subjected to immunoprecipitation with Ab-6KTF-N or Ab-TF-C followed by SDS-PAGE, and exposed on x-ray film for 3 weeks. Equal amounts of transfecting RNA and cells were used for each sample.

Peptide sequences for the 6K and TF proteins for representative alphavirus sequences. The frameshift site (amino acids 'FL', except in BEBV) is shown in bold. For BEBV, which lacks the U UUU UUA motif, the approximate location of the presumed frameshift was determined by alignment to the other sequences. '|'s represent the E2-6K and 6K-E1 cleavage sites and '*'s represent the TF protein termination codon.

Virus-specific detection of SFV 6K and TF proteins. Lanes 1–3: total lysate from SFV-infected (WT; 1 hr and overnight, o/n) and non-infected (-) cells. Lanes 4–6: virions purified from the media (WT) and mock purified virions from non-infected cells (-). Equal amounts of transfecting RNA and cells were used for each sample. All lanes are from the same gel – exposed on x-ray film for 2 weeks to enhance the faint bands corresponding to any low molecular mass products.

Immunofluorescence of SFV-infected cells showing location of 6K and TF proteins. Green fluorescence indicates Abs binding to target peptides. Cell nuclei are stained blue. Cells fixed in acetone are permeabilized, allowing intracellular Ab staining. Cells fixed in 4% PFA are not permeabilized, thus only allowing Abs to bind to peptides at the cell surface. Cells were infected with WT SFV4 virus (WT), the TF knockout mutant (TF^-), or are non-infected controls (-). TF^- serves as an additional control for Ab-TF-C. See also Additional file .

Detection of SFV 6K and/or TF proteins for WT and TF^- viruses. (A) SFV-infected cells were labelled with [³⁵S]Met/Cys and cell lysates (1 hr and overnight, o/n) and purified virions were analyzed by SDS-PAGE. Equal amounts of transfecting RNA and cells were used for each sample. Lanes 1–4 and 7–8 are from the same gel, lanes 5–6 are from a separate gel; Phospho-Imager, 2 days exposure. Negative controls are shown in Figure 8. (B) As above, but with higher sample loading.

Nucleotide and amino acid sequences for 6K and TF in SFV. (A) Nucleotide sequence for 6K and flanking regions, with the polyprotein and -1 frame amino acid sequences given below. The cleavage sites between E1, E2 and 6K are marked. Also marked are the frameshift site U UUU UUA, the TF termination codon, and the position of the point mutation used for the knockout mutant TF^-. (B) Amino acid sequences for the 6K and TF proteins. Three antigens against which three separate Abs were raised are marked by underscores. Peptides with clear mass spectrometry detections are marked by overscores.

Potential stimulatory RNA secondary structures for -1 frameshifting in representative alphavirus species. Stems marked as 'potential' were not supported by dual luciferase mutational analyses (B Chung et al, in preparation), though it is possible that they may still be important in the context of the full 26S sgRNA in virus-infected cells. Viruses: Seal louse (SESV) – [GenBank:AF315122]; Middelburg (MIDV) – [GenBank:AF339486]; Venezuelan equine encephalitis (VEEV) – [GenBank:NC_001449]; Ndumu (NDUV) – [GenBank:AF339487]; Sindbis (SINV) – [GenBank:NC_001547]; Barmah Forest (BFV) – [GenBank:NC_001786]; Sleeping disease (SDV) – [GenBank:NC_003433]; Eastern equine encephalitis (EEEV) – [GenBank:NC_003899].

Phylogenetic nucleotide conservation plots for selected alphavirus within-species full-genome sequence alignments. The nucleotide conservation in a 51-nt sliding window is expressed as a p-value plot, giving the probability that the conservation in the window would be as great or greater than that observed, if a given null model (CDS annotation) was true. Here the null model was set to 'non-coding' in order to give a straightforward nucleotide conservation plot. Plots are given for alignments of (1a) 7 Sindbis virus (SINV) sequences, (2a) 9 Eastern equine encephalitis virus (EEEV) sequences, (3a) 22 Venezuelan equine encephalitis virus (VEEV) sequences, and (4a) 19 Chikungunya virus (CHIKV) sequences. Panels (1-4b) show the phylogenetically summed sequence divergence (mean number of base variations per nucleotide column) for the sequences that contribute to the statistics at each position in the alignment. In any particular column, some sequences may be omitted from the statistical calculations due to alignment gaps. Statistics in regions with lower summed divergence (i.e. partially gapped regions) have a lower signal-to-noise ratio and/or may be omitted from the plot. Panels (1-4c) show the location of the non-structural (CDS1; green) and structural (CDS2; green) CDSs, the non-coding regions (black), and the location of the overlapping -1 frame ORF (red), in the GenBank RefSeqs NC_001547 (SINV), NC_003899 (EEEV), NC_001449 (VEEV) and NC_004162 (CHIKV). The location of the U UUU UUA motif coincides with the 5' end of this ORF. Plots were produced with the CDS-plotcon webserver (Firth, unpublished).

Potential downstream RNA secondary structures in all sequences analysed. (Continued in Figure 4.) As of 20 April 2008, there were 357 alphavirus sequences in GenBank with coverage of the U UUU UUA motif in the 6K cistron. The 100 nt region starting from the U UUU UUA motif, and including the first 93 nt of 3'-adjacent sequence, was extracted from all 357 sequences (although in 26 sequences a shorter region had to be used due to incomplete sequence data). Shown here are the 108 unique ≤100-nt sequences, plus an additional seven duplicate sequences also included since they have different species/strain annotations. The total number of duplicate sequences represented by each sequence shown is given in column 1, while column 2 gives an example GenBank accession number for the sequence, and column 3 gives the virus name abbreviation. Potential RNA secondary structures were identified using a combination of RNAfold and alidot [], pknots [], and manual inspection. Bases within potential stems are indicated either in colour or with underlines (if overlapping other potential stems) and potential base-pairings are indicated with brackets – '()', '[]' or '<>'. '<>' signify more dubious base-pairings, including stems that were experimentally shown not to affect frameshifting efficiency (dual luciferase assays with inserts comprising the U UUU UUA motif and 3'-adjacent sequence; B Chung et al, in preparation). Base variations that maintain base-pairings are marked in bold. Note that not all sequences in GenBank represent functional (infectious) viruses and it is possible that certain sequences whose shift site and/or predicted RNA structure do not conform with the majority of isolates for the same species may represent defective viruses – for example the non-standard slippery heptanucleotide in the SPDV sequence AJ012631 is due to a 36-codon deletion in 6K relative to other SPDV sequences.