Molecular archeological evidence in support of the repeated loss of a papillomavirus gene

Abstract

It is becoming clear that, in addition to gene gain, the loss of genes may be an important evolutionary mechanism for many organisms. However, gene loss is often associated with an increased mutation rate, thus quickly erasing evidence from the genome. The analysis of evolutionarily related sequences can provide empirical evidence for gene loss events. This paper analyzes the sequences of over 300 genetically distinct papillomaviruses and provides evidence for a role of gene loss during the evolution of certain papillomavirus genomes. Phylogenetic analysis suggests that the viral E6 gene was lost at least twice. Despite belonging to distant papillomaviral genera, these viruses lacking a canonical E6 protein may potentially encode a highly hydrophobic protein from an overlapping open reading frame, which we designate E10. Evolutionary pressure working on this alternative frame, may explain why, despite having lost the E6 open reading frame between 20 and 60 million years ago, evidence of an E6-like protein is conserved.

Figure 1. Maximum likelihood phylogenetic tree representing the evolutionary relationships among the Papillomaviridae.

Maximum likelihood phylogenetic tree of the Papillomaviridae. This tree is based on a partitioned supermatrix derived from the E1, E2, L2 and L1 nucleotide sequences. Viruses indicated with a red clade do not contain a canonical E6 ORF, purple clades identify the avian/turtle viruses that contain a “single domain” E6 protein. Classification was based on. The asterisk (*) indicates that some members of this clade have not yet been officially recognized as members of the Gammapapillomavirus genus. Colored nodes indicate bootstrap support (red = 100%, green >90%, blue >75%). The red triangle indicates the MRCA of Xi- and Gamma papillomaviruses. The arrowhead highlights CPV2, which was chosen to root the phylogenetic tree in Fig. 2.

Figure 2. Time calibrated phylogenetic tree of Gammapapillomavirus sequences.

The evolutionary rate estimated for feline PVs was used to estimate the divergence time of the indicated nodes. Classification was based on. The asterisk (*) indicates that some members of this clade have not yet been officially recognized as members of the Gammapapilliomavirus genus. Support values and exact estimates (with 95% highest posterior density) for each node are presented in supplementary Table 2. The Gamma-6 species is highlighted in red. The tree and geological column were generated using the APE, phyloch (available from http://www.christophheibl.de/Rpackages.html), and strap packages within R. The scale bar indicates millions of years before the present. The Gammapapillomavirus genus tree was rooted on CPV2 (indicated by arrowhead in Fig. 1).

Figure 3. Viruses within the Gammapapillomavirus 6 species contain a putative E5 like open reading frame.

(A) Evidence of a C-terminal deletion within the E6 protein of the Gamma-6 viruses. Alignment of the sequence from the promoter proximal E2 binding site to the stop codon of E7 extracted from Gamma-6 and Gamma-7 papillomaviruses. The phylogenetic tree indicates the evolutionary history, with the black circle highlighting the Gamma-6 species MRCA. Colors indicate nucleotides (A = red; T = green, C = blue, G = yellow). Supplemental Figure 1 has a base pair level version of this alignment. The purple bar indicates the E6 ORF (alignment in 3B), red and green bars mark the putative E10 and E7 ORFs (E10 alignment in 3C). (B) Sequence alignment of the putative gamma-6 E6 remnant and related Gammapapillomavirus E6 proteins. The alignment is shaded by conservation. Red asterisks (*) indicate internal STOP codons. Zinc-binding motifs are indicated below the alignment. The phylogenetic tree indicates the evolutionary history, with the black circle highlighting the Gamma-6 species MRCA. (C) Sequence alignment of the putative gamma-6 E10 protein and related Gammapapillomavirus proteins. The alignment is shaded by conservation. Red asterisks (*) indicate internal STOP codons The phylogenetic tree indicates the evolutionary history, with the black circle highlighting the Gamma-6 species MRCA. (D) The putative promoter of E6-minus viruses is located in the correct syntenic environment. Sequence alignment of the DNA sequence upstream of the putative E6 start codon. A consensus E2 binding site, putative TATA box, and E6 start codon are indicated. A sequence logo shows the consensus sequence. The phylogenetic tree indicates the evolutionary history, with the black circle highlighting the MRCA of the Gamma-6 species. (E) Hydropathicity scale of the Delta E5, Xi E10, and Gamma-6 E10-like proteins. Kyte – Doolittle and Hopp – Woods protein scales were calculated using a sliding window with size of 19 aa. Values were calculated for individual E5-like proteins (Delta; n = 13, Xi; n = 9, and Gamma-6; n = 5). The graph shows the average value for each group. The first 24 windows for the Delta E5 proteins are shown. For the Kyte – Doolittle graphs, values greater than 1.8 (indicated by the dotted line) represent possible transmembrane regions.