A potentially novel overlapping gene in the genomes of Israeli acute paralysis virus and its relatives
© Sabath et al; licensee BioMed Central Ltd. 2009
Received: 2 July 2009
Accepted: 17 September 2009
Published: 17 September 2009
The Israeli acute paralysis virus (IAPV) is a honeybee-infecting virus that was found to be associated with colony collapse disorder. The IAPV genome contains two genes encoding a structural and a nonstructural polyprotein. We applied a recently developed method for the estimation of selection in overlapping genes to detect purifying selection and, hence, functionality. We provide evolutionary evidence for the existence of a functional overlapping gene, which is translated in the +1 reading frame of the structural polyprotein gene. Conserved orthologs of this putative gene, which we provisionally call pog (p redicted o verlapping g ene), were also found in the genomes of a monophyletic clade of dicistroviruses that includes IAPV, acute bee paralysis virus, Kashmir bee virus, and Solenopsis invicta (red imported fire ant) virus 1.
Colony collapse disorder (CCD) is a syndrome characterized by the mass disappearance of honeybees from hives . CCD imperils a global resource estimated at approximately $200 billion . For example, it has been estimated that up to 35% of hives in the US may have been affected . Many culprits have been suggested as causal factors of CCD, among them fungal, bacterial, and protozoan diseases, external and internal parasites, in-hive chemicals, agricultural insecticides, genetically modified crops, climatic factors, changed cultural practices, and the spread of cellular phones . The Israeli acute paralysis virus (IAPV), a positive-strand RNA virus belonging to the family Dicistroviridae, was found to be strongly correlated with CCD . It was first isolated in Israel , but was later found to have a worldwide distribution [4, 6, 7].
The genome of IAPV contains two long open reading frames (ORFs) separated by an intergenic region. The 5' ORF encodes a structural polyprotein; the 3' ORF encodes a non-structural polyprotein . The non-structural polyprotein contains several signature sequences for helicase, protease, and RNA-dependent RNA polymerase . The structural polyprotein, which is located downstream of the non-structural polyprotein, encodes two (and possibly more) capsid proteins.
Overlapping genes are easily missed by annotation programs , as evidenced by the fact that several overlapping genes were only detected by using the signatures of purifying selection [9–13]. Here, we apply a recently developed method for the detection of selection in overlapping reading frames  to the genome of IAPV and its relatives.
Results and Discussion
A list of completely sequenced dicistroviruses used in this study
Israel acute paralysis virus (IAPV)
Acute bee paralysis virus (ABPV)
Kashmir bee virus (KBV)
Solenopsis invicta virus (SINV-1)
Black queen cell virus (BQCV)
Cricket paralysis virus (CrPV)
Homalodisca coagulata virus-1 (HoCV-1)
Drosophila C virus (DCV)
Aphid lethal paralysis virus (ALPV)
Himetobi P virus (HiPV)
Taura syndrome virus (TSV)
Plautia stali intestine virus (PSIV)
Triatoma virus (TrV)
Rhopalosiphum padi virus (RhPV)
Clusters of orthologous overlapping ORFs on the positive strand
Start of ORF
End of ORF
Sequence conservation in comparisons of known orthologous proteins and orthologous products of overlapping ORFs.
Identity of known proteins (%)
Identity of hypothetical product of overlapping ORFs (%)
An additional indication for selection on these ORFs was obtained by comparing the degrees of conservation of the hypothetical protein sequences of the overlapping ORFs against the protein sequences of the known genes (structural and nonstructural polyproteins, Table 3). The degree of amino-acid conservation and, hence, sequence identity between orthologous protein-coding genes is influenced ceteris paribus by the intensity of purifying selection. If both overlapping genes are under similar strengths of selection, the amino-acid sequence identity of one pair of homologous genes would be similar to that of the overlapping pair. On the other hand, if a functional gene overlaps a non-functional ORF, the amino-acid identity between the hypothetical protein sequences of the non-functional ORFs would be much lower than that between the two homologous overlapping functional genes. We found that the degree of amino-acid conservation of the overlapping sequence identity between pairs of overlapping ORFs in cluster A is only slightly lower than that of the known gene (maximum of 12% difference between IAPV and SINV-1 in cluster A, Table 3). In contrast, the amino-acid sequence identity between ORF pairs in clusters B and C is much lower than that between the pairs of known genes (maximum of 44% difference between CrPV and DCV in cluster C, Table 3).
The signature of purifying selection on the ORFs in cluster A suggests that they may encode functional proteins. We provisionally term this gene pog (p redicted o verlapping g ene). In Figure 1, we show that pog is found in the genomes of four viruses that constitute a monophyletic clade, but not in any other dicistrovirid genome (Figure 1A). Its phylogenetic distribution suggests that pog originated before the divergence of SINV-1 from the three bee viruses. The phylogenetic distributions of the ORFs in clusters B and C (Figure 1B) are patchy. This patchiness is an additional indication that the overlapping ORFs in clusters B and C are spurious, i.e., non-functional.
A protein motif search resulted in several matches, all with a weak score. Two patterns were found in all four proteins: (1) a signature of rhodopsin-like GPCRs (G protein-coupled receptors), and (2) a protein kinase C phosphorylation site (Figure 3). Prediction of the secondary structures  suggests that the proteins contain two conserved helix domains, separated by 3-5 residues (except for SINV-1, in which one long domain is predicted), at the C-terminus (Figure 3). A search for transmembrane topology  indicates that the longer helix may be a transmembranal segment (Figure 3). Although viruses often use GPCRs to exploit the host immune system through molecular mimicry [22–25], the lengths of the proteins encoded by pog are shorter than the average virus-encoded GPCR. Therefore, these proteins may have a different function.
In this note, we provide evolutionary evidence (purifying selection) for the existence of a functional overlapping gene, pog, in the genomes of IAPV, ABPV, KBV, and SINV-1. To our knowledge, this putative gene, whose coding region overlaps the structural polyprotein, has not been described in the literature before.
Sequence Data, Processing, and Analysis
Fourteen completely sequenced dicistrovirid genomes were obtained from NCBI (Table 1). Each genome was scanned for the presence of overlapping ORFs. We used BLASTP  with the protein sequences of the known genes to identify matches of orthologous overlapping ORFs (E value < 10-6). Matching overlapping ORFs were assigned into clusters. Within each cluster, we aligned the amino-acid orthologs by using the sequences of the known genes as references. If alignment length of the overlapping sequence exceeded 60 amino-acids, and if the amino-acid sequence identity among the hypothetical genes within a cluster was higher than 65%, we tested for selection on the hypothetical gene (see below).
We aligned the protein sequences of the two polyproteins with CLUSTAW  as implemented in the MEGA package . Alignment quality was confirmed using HoT . We reconstructed two phylogenetic trees (one for each polyprotein) by applying the neighbor joining method , as implemented in the MEGA package . Trees were rooted by the mid-point rooting method  and confidence of each branch was estimated by bootstrap with 1000 replications.
Detection of Selection in Overlapping Genes
We used the method of Sabath et al.  for the simultaneous estimation of selection intensities in overlapping genes. This method uses a maximum-likelihood framework to fit a Markov model of codon substitution to data from two aligned homologous overlapping sequences. To predict functionality of an ORF that overlaps a known gene, we modified an existing approach for predicting functionality in non-overlapping genes . Given two aligned orthologous overlapping sequences, we estimate the likelihood of two hierarchical models. In model 1, there is no selection on the ORF. In model 2, the ORF is assumed to be under selection. The likelihood-ratio test is used to test whether model 2 fits the data significantly better than model 1, in which case, the ORF is predicted to be under selection and most probably functional.
We looked for motifs within the inferred protein sequences encoded by the overlapping ORF by using the motif search server http://motif.genome.jp/ and the My-Hits server http://hits.isb-sib.ch/cgi-bin/PFSCAN with the following motif databases: PRINTS , PROSITE , and Pfam . We used PSIPRED  to predict secondary structure, and MEMSAT  to predict transmembrane protein topology.
Overlapping ORFs on the negative strand
In the fourteen completely sequenced dicistroviruse genomes (Table 1), we identified 240 overlapping ORFs of length equal or greater than 60 codons on the negative strand. Of the 240 ORFs, 113 were found in concordant genomic locations in two or more genomes. The concordant overlapping ORFs were assigned into 29 clusters (Additional file 1). There are 9, 1, and 19 clusters in phase 0, 1, and 2, respectively. The cluster size ranges from 2 to 9. In two clusters, 5 and 10, both in phase 2, there is a weak signature of selection. However, this signature seems to be a false positive, which was driven by the unique structure of opposite-strand phase-2 overlap (Additional file 2). In this structure, codon positions one and two of one gene match codon positions two and one of the overlapping gene. This structure leads to a situation where most changes are either synonymous or nonsynonymous in both overlapping genes and occasionally, to false signal of purifying selection on the overlapping ORF. In addition, one of the clusters (cluster 10) does not constitute a monophyletic clade, and is, therefore, unlikely to be functional. We therefore conclude that dicistroviruses most probably do not encode proteins on the negative strand.
We thank Dr. Ilan Sela and an anonymous reviewer for their comments. This work was supported in part by US National Library of Medicine Grant LM010009-01 to Dan Graur and Giddy Landan and by the Small Grants Program of the University of Houston.
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