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Bioinformatic analysis suggests that the Orbivirus VP6 cistron encodes an overlapping gene.

Firth AE - Virol. J. (2008)

Bottom Line: Again, the pattern of base variations across sequence alignments indicates multiple coding in the VP6 and ORFX reading frames.At approximately 9.5 kDa, the putative ORFX product in BTV is too small to appear on most published protein gels.We hope that presentation of this bioinformatic analysis will stimulate an attempt to experimentally verify the expression and functional role of ORFX, and hence lead to a greater understanding of the molecular biology of these important pathogens.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, BioSciences Institute, University College Cork, Cork, Ireland. A.Firth@ucc.ie

ABSTRACT

Background: The genus Orbivirus includes several species that infect livestock - including Bluetongue virus (BTV) and African horse sickness virus (AHSV). These viruses have linear dsRNA genomes divided into ten segments, all of which have previously been assumed to be monocistronic.

Results: Bioinformatic evidence is presented for a short overlapping coding sequence (CDS) in the Orbivirus genome segment 9, overlapping the VP6 cistron in the +1 reading frame. In BTV, a 77-79 codon AUG-initiated open reading frame (hereafter ORFX) is present in all 48 segment 9 sequences analysed. The pattern of base variations across the 48-sequence alignment indicates that ORFX is subject to functional constraints at the amino acid level (even when the constraints due to coding in the overlapping VP6 reading frame are taken into account; MLOGD software). In fact the translated ORFX shows greater amino acid conservation than the overlapping region of VP6. The ORFX AUG codon has a strong Kozak context in all 48 sequences. Each has only one or two upstream AUG codons, always in the VP6 reading frame, and (with a single exception) always with weak or medium Kozak context. Thus, in BTV, ORFX may be translated via leaky scanning. A long (83-169 codon) ORF is present in a corresponding location and reading frame in all other Orbivirus species analysed except Saint Croix River virus (SCRV; the most divergent). Again, the pattern of base variations across sequence alignments indicates multiple coding in the VP6 and ORFX reading frames.

Conclusion: At approximately 9.5 kDa, the putative ORFX product in BTV is too small to appear on most published protein gels. Nonetheless, a review of past literature reveals a number of possible detections. We hope that presentation of this bioinformatic analysis will stimulate an attempt to experimentally verify the expression and functional role of ORFX, and hence lead to a greater understanding of the molecular biology of these important pathogens.

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MLOGD statistics for BTV, AHSV, PALV and PHSV/YUOV alignments. Output plots from MLOGD used in the 'Test Query CDS' mode, applied to the ORFX region in BTV, AHSV, PALV and PHSV/YUOV sequence alignments. See [16] for full details of the MLOGD software. The  model comprises the VP6 CDS and the query CDS is ORFX. In each plot, the top panel displays the raw log(LR) statistics at each alignment position. There is a separate track for each reference – non-reference sequence pair (labelled at the right, together with the pairwise divergences; albeit not legible for the BTV alignment since it contains so many – i.e. 48 – sequences). Stop codons (of which there are none except 3' terminal ones) in each of the VP6 and ORFX reading frames, and alignment gaps for each sequence, are marked on the appropriate tracks. The second panel displays the Σtree log(LR) statistic at each alignment position, where 'tree' represents a phylogenetic tree – see [16]. The third and fourth panels display sliding window means of the statistics in the first and second panels, respectively. The fifth panel shows the locations of the  and alternative model CDSs (i.e. VP6 and ORFX, respectively). The sixth panel shows the summed mean sequence divergence (base variations per alignment nt column) for the sequence pairs that contribute to the Σtree log(LR) statistic at each alignment position. This is a measure of the information available at each alignment position (e.g. partially gapped regions have lower summed mean sequence divergence). The predominantly positive values in the fourth panel indicate that ORFX is subject to functional constraints, at the amino acid level, over the majority of its length.
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Figure 3: MLOGD statistics for BTV, AHSV, PALV and PHSV/YUOV alignments. Output plots from MLOGD used in the 'Test Query CDS' mode, applied to the ORFX region in BTV, AHSV, PALV and PHSV/YUOV sequence alignments. See [16] for full details of the MLOGD software. The model comprises the VP6 CDS and the query CDS is ORFX. In each plot, the top panel displays the raw log(LR) statistics at each alignment position. There is a separate track for each reference – non-reference sequence pair (labelled at the right, together with the pairwise divergences; albeit not legible for the BTV alignment since it contains so many – i.e. 48 – sequences). Stop codons (of which there are none except 3' terminal ones) in each of the VP6 and ORFX reading frames, and alignment gaps for each sequence, are marked on the appropriate tracks. The second panel displays the Σtree log(LR) statistic at each alignment position, where 'tree' represents a phylogenetic tree – see [16]. The third and fourth panels display sliding window means of the statistics in the first and second panels, respectively. The fifth panel shows the locations of the and alternative model CDSs (i.e. VP6 and ORFX, respectively). The sixth panel shows the summed mean sequence divergence (base variations per alignment nt column) for the sequence pairs that contribute to the Σtree log(LR) statistic at each alignment position. This is a measure of the information available at each alignment position (e.g. partially gapped regions have lower summed mean sequence divergence). The predominantly positive values in the fourth panel indicate that ORFX is subject to functional constraints, at the amino acid level, over the majority of its length.

Mentions: The putative new CDS, ORFX, was first identified in a BTV sequence alignment, using MLOGD. In the RefSeq [GenBank: NC_006008] (1049 nt), ORFX has coords 182..415 (77 codons) and therefore is completely contained within the VP6 cistron (16..1005), overlapping it in the +1 reading frame (Figure 1). When applied to an alignment of 48 BTV sequences (see Methods; pairwise divergences ≤0.21 base variations per nucleotide and total alignment divergence ~0.77 independent base variations per column in the ORFX region), MLOGD detected a strong coding signature for ORFX (Figures 2, 3). There are ~180 independent base variations across the alignment in the ORFX region, thus providing MLOGD with a robust signal. Formally, and within the MLOGD model, p < 10-40. Indeed Figure 2 shows four non-overlapping – and hence completely independent – positively scoring windows in the ORFX region. Moreover, the MLOGD results showed that, within the ORFX region, ORFX is more conserved at the amino acid level than VP6 (Figure 2). Finally, inspection of the MLOGD output showed that the ORF is present in all of the 48 sequences (i.e. no premature termination codons; Figure 2).


Bioinformatic analysis suggests that the Orbivirus VP6 cistron encodes an overlapping gene.

Firth AE - Virol. J. (2008)

MLOGD statistics for BTV, AHSV, PALV and PHSV/YUOV alignments. Output plots from MLOGD used in the 'Test Query CDS' mode, applied to the ORFX region in BTV, AHSV, PALV and PHSV/YUOV sequence alignments. See [16] for full details of the MLOGD software. The  model comprises the VP6 CDS and the query CDS is ORFX. In each plot, the top panel displays the raw log(LR) statistics at each alignment position. There is a separate track for each reference – non-reference sequence pair (labelled at the right, together with the pairwise divergences; albeit not legible for the BTV alignment since it contains so many – i.e. 48 – sequences). Stop codons (of which there are none except 3' terminal ones) in each of the VP6 and ORFX reading frames, and alignment gaps for each sequence, are marked on the appropriate tracks. The second panel displays the Σtree log(LR) statistic at each alignment position, where 'tree' represents a phylogenetic tree – see [16]. The third and fourth panels display sliding window means of the statistics in the first and second panels, respectively. The fifth panel shows the locations of the  and alternative model CDSs (i.e. VP6 and ORFX, respectively). The sixth panel shows the summed mean sequence divergence (base variations per alignment nt column) for the sequence pairs that contribute to the Σtree log(LR) statistic at each alignment position. This is a measure of the information available at each alignment position (e.g. partially gapped regions have lower summed mean sequence divergence). The predominantly positive values in the fourth panel indicate that ORFX is subject to functional constraints, at the amino acid level, over the majority of its length.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 3: MLOGD statistics for BTV, AHSV, PALV and PHSV/YUOV alignments. Output plots from MLOGD used in the 'Test Query CDS' mode, applied to the ORFX region in BTV, AHSV, PALV and PHSV/YUOV sequence alignments. See [16] for full details of the MLOGD software. The model comprises the VP6 CDS and the query CDS is ORFX. In each plot, the top panel displays the raw log(LR) statistics at each alignment position. There is a separate track for each reference – non-reference sequence pair (labelled at the right, together with the pairwise divergences; albeit not legible for the BTV alignment since it contains so many – i.e. 48 – sequences). Stop codons (of which there are none except 3' terminal ones) in each of the VP6 and ORFX reading frames, and alignment gaps for each sequence, are marked on the appropriate tracks. The second panel displays the Σtree log(LR) statistic at each alignment position, where 'tree' represents a phylogenetic tree – see [16]. The third and fourth panels display sliding window means of the statistics in the first and second panels, respectively. The fifth panel shows the locations of the and alternative model CDSs (i.e. VP6 and ORFX, respectively). The sixth panel shows the summed mean sequence divergence (base variations per alignment nt column) for the sequence pairs that contribute to the Σtree log(LR) statistic at each alignment position. This is a measure of the information available at each alignment position (e.g. partially gapped regions have lower summed mean sequence divergence). The predominantly positive values in the fourth panel indicate that ORFX is subject to functional constraints, at the amino acid level, over the majority of its length.
Mentions: The putative new CDS, ORFX, was first identified in a BTV sequence alignment, using MLOGD. In the RefSeq [GenBank: NC_006008] (1049 nt), ORFX has coords 182..415 (77 codons) and therefore is completely contained within the VP6 cistron (16..1005), overlapping it in the +1 reading frame (Figure 1). When applied to an alignment of 48 BTV sequences (see Methods; pairwise divergences ≤0.21 base variations per nucleotide and total alignment divergence ~0.77 independent base variations per column in the ORFX region), MLOGD detected a strong coding signature for ORFX (Figures 2, 3). There are ~180 independent base variations across the alignment in the ORFX region, thus providing MLOGD with a robust signal. Formally, and within the MLOGD model, p < 10-40. Indeed Figure 2 shows four non-overlapping – and hence completely independent – positively scoring windows in the ORFX region. Moreover, the MLOGD results showed that, within the ORFX region, ORFX is more conserved at the amino acid level than VP6 (Figure 2). Finally, inspection of the MLOGD output showed that the ORF is present in all of the 48 sequences (i.e. no premature termination codons; Figure 2).

Bottom Line: Again, the pattern of base variations across sequence alignments indicates multiple coding in the VP6 and ORFX reading frames.At approximately 9.5 kDa, the putative ORFX product in BTV is too small to appear on most published protein gels.We hope that presentation of this bioinformatic analysis will stimulate an attempt to experimentally verify the expression and functional role of ORFX, and hence lead to a greater understanding of the molecular biology of these important pathogens.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, BioSciences Institute, University College Cork, Cork, Ireland. A.Firth@ucc.ie

ABSTRACT

Background: The genus Orbivirus includes several species that infect livestock - including Bluetongue virus (BTV) and African horse sickness virus (AHSV). These viruses have linear dsRNA genomes divided into ten segments, all of which have previously been assumed to be monocistronic.

Results: Bioinformatic evidence is presented for a short overlapping coding sequence (CDS) in the Orbivirus genome segment 9, overlapping the VP6 cistron in the +1 reading frame. In BTV, a 77-79 codon AUG-initiated open reading frame (hereafter ORFX) is present in all 48 segment 9 sequences analysed. The pattern of base variations across the 48-sequence alignment indicates that ORFX is subject to functional constraints at the amino acid level (even when the constraints due to coding in the overlapping VP6 reading frame are taken into account; MLOGD software). In fact the translated ORFX shows greater amino acid conservation than the overlapping region of VP6. The ORFX AUG codon has a strong Kozak context in all 48 sequences. Each has only one or two upstream AUG codons, always in the VP6 reading frame, and (with a single exception) always with weak or medium Kozak context. Thus, in BTV, ORFX may be translated via leaky scanning. A long (83-169 codon) ORF is present in a corresponding location and reading frame in all other Orbivirus species analysed except Saint Croix River virus (SCRV; the most divergent). Again, the pattern of base variations across sequence alignments indicates multiple coding in the VP6 and ORFX reading frames.

Conclusion: At approximately 9.5 kDa, the putative ORFX product in BTV is too small to appear on most published protein gels. Nonetheless, a review of past literature reveals a number of possible detections. We hope that presentation of this bioinformatic analysis will stimulate an attempt to experimentally verify the expression and functional role of ORFX, and hence lead to a greater understanding of the molecular biology of these important pathogens.

Show MeSH
Related in: MedlinePlus