Evolution of Viral Proteins Originated De Novo by Overprinting

Sabath, Niv; Wagner, Andreas; Karlin, David

To identify de novo gene candidates, we applied the criterion of monophyly stated in the introduction: one of the genes in an overlapping pair-the ancestral gene-must occur in each member of a viral clade, whereas the other gene-the de novo gene-must be restricted to a single subclade, the focal clade ( fig. 1b) . To find genes that meet this criterion, we first identified, for each gene pair, homologous protein products in related genomes. (We found no evidence of duplicated genes in the genomes under study, hence all our homologs are orthologs.) We aligned the homologous protein sequences of the ancestral protein (which is more phylogenetically widespread) and constructed their phylogenetic tree. By manual examination of these trees, we identified 12 cases of de novo origin (tables 1 and 3) that met the criterion of monophyly. We scanned the related genomes of the focal clade for unannotated ORFs to overcome missing genes due to fault annotation. These trees also allowed us to infer the internal node of a tree closest to the origin of the de novo gene. We call this node the last common ancestor (LCA, marked with a blue circle in the hypothetical example of fig. 1b ). To ensure that the identification of the LCA is not biased by genome annotation, we manually examined the related genomes for presence of homologous unannotated ORFs. We provide detailed explanations of the challenges in de novo gene and LCA identification in the supplementary information, Supplementary Material online (supplementary figs. S1-S4, Supplementary Material online). The phylogenetic tree and the corresponding genomic maps of the 12 cases are presented in supplementary figure S5, Supplementary Material online. For all gene pairs that met the monophyly criterion, we also determined the DNA sequence alignments corresponding to the amino acid sequence alignments (of the ancestral proteins), to enable the calculations described later.