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. 2009 Dec 17:6:222.
doi: 10.1186/1743-422X-6-222.

Evidence for a novel coding sequence overlapping the 5'-terminal approximately 90 codons of the gill-associated and yellow head okavirus envelope glycoprotein gene

Andrew E Firth  1 , John F Atkins
Affiliations

Evidence for a novel coding sequence overlapping the 5'-terminal approximately 90 codons of the gill-associated and yellow head okavirus envelope glycoprotein gene

Andrew E Firth et al. Virol J. .

Abstract

The genus Okavirus (order Nidovirales) includes a number of viruses that infect crustaceans, causing major losses in the shrimp industry. These viruses have a linear positive-sense ssRNA genome of approximately 26-27 kb, encoding a large replicase polyprotein that is expressed from the genomic RNA, and several additional proteins that are expressed from a nested set of 3'-coterminal subgenomic RNAs. In this brief report, we describe the bioinformatic discovery of a new, apparently coding, ORF that overlaps the 5' end of the envelope glycoprotein encoding sequence, ORF3, in the +2 reading frame. The new ORF has a strong coding signature and, in fact, is more conserved at the amino acid level than the overlapping region of ORF3. We propose that translation of the new ORF initiates at a conserved AUG codon separated by just 2 nt from the ORF3 AUG initiation codon, resulting in a novel 86 amino acid protein.

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Figures

Figure 1
Figure 1
Coding potential statistics for okavirus ORF3 and the overlapping ORFX. (A) Okavirus genome map (GAV [GenBank:AF227196]). (B2-B10) Coding potential statistics based on an alignment of seven okavirus full-length ORF3 sequences (see text for accession numbers). (B2-B4) Positions of stop codons in each of the three forward reading frames. Note the conserved absence of stop codons in the +2 frame within ORFX. (B5-B6) Conservation at synonymous sites within ORF3 (see [15]). (B5) depicts the probability that the degree of conservation within a given window could be obtained under a null model of neutral evolution at synonymous sites, while (B6) depicts the ratio of the observed number of substitutions within a given window to the number expected under the null model. (B7-B9) MLOGD sliding-window plots (see [12]). The null model, in each window, is that the sequence is non-coding, while the alternative model is that the sequence is coding in the given reading frame. Positive scores favour the alternative model and, as expected, there is a strong coding signature in the +0 frame (B7) throughout ORF3 except where ORF3 is overlapped by ORFX. In the +1 and +2 frames (B8-B9), scores are generally negative. However, the ORFX region has consecutive high positively scoring windows (B9). (B10) MLOGD statistics restricted to ORFX. Here, for increased sensitivity, the null and alternative models were fitted specifically for the ORFX region. The null model is that only the ORF3 frame is coding, while the alternative model is that both the ORF3 frame and ORFX are coding.
Figure 2
Figure 2
Nucleotide and amino acid sequence alignments. (A) Nucleotide alignment of the 5'-terminal region of the ORF3 sgRNA. All currently available and non-identical okavirus sequences with coverage of this region are shown. A further four sequences (DQ978360, FJ194949, FJ848673 and EF156405) are locally identical to EU487200. Genotype designations within the Yellow head complex are based on the ORF3 phylogeny of Ref. [18]. Spaces separate +0/ORF3-frame codons. All AUG codons are indicated in capitals. Colour coding is as follows: light blue - ORF3 initiation codon(s); green - proposed +2/ORFX-frame initiation codon; dark blue - upstream +1 frame AUG codon (31 codon ORF); orange - last upstream ORFX-frame stop codons. (B) Amino acid alignment of the translated ORFX for representative okavirus sequences.
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