- Short report
- Open Access
Analysis of the coding potential of the partially overlapping 3' ORF in segment 5 of the plant fijiviruses
© Firth and Atkins; licensee BioMed Central Ltd. 2009
Received: 26 February 2009
Accepted: 17 March 2009
Published: 17 March 2009
The plant-infecting members of the genus Fijivirus (family Reoviridae) have linear dsRNA genomes divided into 10 segments, two of which contain two substantial and non-overlapping ORFs, while the remaining eight are apparently monocistronic. However, one of these – namely segment 5 – contains a second long ORF (~200+ codons) that overlaps the 3' end of the major ORF (~920–940 codons) in the +1 reading frame. In this report, we use bioinformatic techniques to analyze the pattern of base variations across an alignment of fijivirus segment 5 sequences, and show that this 3' ORF has a strong coding signature. Possible translation mechanisms for this unusually positioned ORF are discussed.
The genus Fijivirus is one of ≥12 genera within the Reoviridae, a family of segmented dsRNA viruses. Fijiviruses have 10 segments, and infect plants and insects. Species such as Fiji disease virus (FDV), Mal de Rio Cuarto virus (MRCV) and Rice black streaked dwarf virus (RBSDV) are transmitted by planthoppers and replicate in both the insect and plant hosts, while the more distantly related Nilaparvata lugens reovirus replicates only in insects. Reovirus segments are predominantly monocistronic. However, two of the plant fijivirus segments (S7 and S9 in RBSDV, homologous segments in other sequenced plant fijiviruses) contain two non-overlapping coding sequences (CDSs), each pair separated by a short non-coding sequence [1, 2]. One other plant fijivirus segment (S5 in RBSDV) contains a second substantial open reading frame or ORF (hereafter ORF5-2; ~200+ codons), that overlaps the 3' end of the 'major' CDS (hereafter ORF5-1; ~920–940 codons) in the +1 reading frame. The presence of this open reading frame has been noted previously in RBSDV [3, 4]. However, it has often been ignored in the fijivirus literature; it is not currently annotated in any of the three GenBank plant fijivirus RefSeqs; and its unusual genomic location means that its coding status remains uncertain without further analysis. In this short report, we present bioinformatic evidence that ORF5-2 is in fact coding, and initiates ≥365 nt before the 3' end of ORF5-1 – thus implying that it utilizes an unusual, as yet undefined, expression mechanism.
Fijivirus sequences in GenBank (9 Feb 2009) homologous to RBSDV segment 5 were located by applying tblastn  to the RBSDV segment 5 RefSeq [GenBank: NC_003736] ORF5-1 amino acid sequence, resulting in the additional sequences [GenBank: AY144569] – RBSDV segment 5, [GenBank: NC_007160] – FDV segment 5, and [GenBank: NC_008735] – MRCV segment 5. Note that the GenBank RefSeqs NC_003736, NC_007160 and NC_008735 were derived, respectively, from AJ409147, AY029521 and AY607587.
If initiation is assumed to occur at the first in-frame AUG codon then, in the RBSDV RefSeq , the two segment 5 ORFs have nucleotide coordinates 16–2826 (ORF5-1; 107 kDa) and 2378–3070 (ORF5-2; 26.7 kDa), so that ORF5-2 has 231 codons and overlaps the 3' end of ORF5-1 by 449 nt in the +1 reading frame. In the other RBSDV sequence (AY144569; ), ORF5-1 has the same nucleotide coordinates (i.e. 16–2826), but the first suitable AUG codon for ORF5-2 is 28 codons further 3', giving ORF5-2 the maximal AUG-initiated nucleotide coordinates 2462–3070 (203 codons; 23.4 kDa; 365 nt overlap). In MRCV , the maximal AUG-initiated nucleotide coordinates are 16–2811 (ORF5-1) and 2366–3130 (ORF5-2), so that ORF5-2 has 255 codons and overlaps the 3' end of ORF5-1 by 446 nt, again in the +1 reading frame.
In the FDV RefSeq, NC_007160, the two overlapping ORFs have apparently been 'merged' into a single long ORF . However, in the expected overlap region, there is a +1 frame overlapping ORF of up to 128 codons that has high amino acid homology to the N-terminal half of ORF5-2 in RBSDV and MRCV. Moreover, the C-terminal ~106 amino acid region of the annotated FDV ORF5-1 has significant homology to the C-terminal half of ORF5-2 in RBSDV and MRCV. This suggests that NC_007160 contains a deletion within or near to the region 2718–2746. Whether this is a real feature of FDV, or whether NC_007160 simply represents a 'defective' sequence, merits further investigation. For the analyses presented here, however, an additional 'U' was arbitrarily inserted into a run of five 'U's at the location 2714–2718 and, in this case, ORF5-1 and ORF5-2 assume respective maximal AUG-initiated nucleotide coordinates 59–2827 and 2448–3065, so that ORF5-2 has 206 codons and overlaps the 3' end of ORF5-1 by 380 nt in the +1 reading frame. (Note that our results do not depend on the FDV sequence – if it is excluded from the analyses, we obtain essentially the same results for the remaining fijivirus sequences.)
The bottom panel of Figure 1 shows a representation of the conservation at synonymous sites in ORF5-1. Beginning with pairwise sequence comparisons, conservation at synonymous sites (only) was evaluated by comparing the observed number of base substitutions with the number expected under a neutral evolution model. The procedure takes into account whether synonymous site codons are 1-, 2-, 3-, 4- or 6-fold degenerate and the differing probabilities of transitions and transversions (full details are available on request from the authors). Statistics were then summed over a phylogenetic tree as described in , and averaged over a sliding window. Peaks in the conservation at synonymous sites are generally indicative of functionally important overlapping elements – including overlapping CDSs – and it can be seen that the highest conservation at synonymous sites in ORF5-1 corresponds to the region where it overlaps ORF5-2.
Further, albeit indirect, evidence for the coding status of ORF5-2 comes from an analysis of the 3'UTR lengths of the 10 segments. In RBSDV, for example, the 3'UTR lengths for segments 1–4 and 6–10 are 71, 86, 117, 74, 185, 81, 136, 111 and 103 nt. If ORF5-2 is not a CDS, then segment 5 has an unusually long 3'UTR (335 nt). However, if ORF5-2 is coding, then the 3'UTR length (91 nt) is within the range of 3' UTR lengths for the other 9 segments.
Since subgenomic RNAs are unknown in the Reoviridae family, the genomic location of ORF5-2 rules out most possible translation mechanisms. In NC_003736 (RBSDV), for example, ORF5-1 potentially begins at AUG1 while ORF5-2 (if AUG-initiated) begins at AUG56 or later, thus precluding conventional leaky scanning . Reinitiation appears unlikely since, given the 5'-extent of the positive MLOGD coding signal, it would appear to necessitate backward scanning of ≥365 nt (cf. ). Transcriptional slippage also appears unlikely as no suitable slippage sites were found and, in contrast to the paramyxovirus 'rule of six' , there is no obvious mechanism in the reoviruses for selective packaging of non-edited transcripts. Ribosomal +1 frameshifting from ORF5-1 into ORF5-2 to produce a fusion protein (in competition with conventional translation of ORF5-1) is one possibility . A second possible mechanism is an IRES – examples of which in other viruses can range from complex RNA secondary structures (which we were not able to identify in fijivirus segment 5, in any convincing manner, with the available sequence data) to simple polypurine tracts [15, 16]. A third possible translation mechanism is some sort of ribosomal shunting. Indeed in another Reoviridae species – Avian reovirus – a novel, as yet not fully understood, scanning-independent ribosome migration mechanism is used to bypass two upstream CDSs in order to translate the 3'-proximal CDS on the tricistronic S1 mRNA .
Although much remains to be discovered about even the known fijivirus proteins, it is important to be aware of the full complement of encoded proteins as early as possible. We hope that presentation of this bioinformatic analysis will help fullfil that goal. Initial verification of ORF5-2 product could be by means of immunoblotting with ORF5-2 specific antibodies, bearing in mind, however, that it may be expressed at relatively low levels.
This work was supported by National Institutes of Health Grant R01 GM079523 and an award from Science Foundation Ireland, both to JFA.
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