Degradation of Human PDZ-Proteins by Human Alphapapillomaviruses Represents an Evolutionary Adaptation to a Novel Cellular Niche

<div><p>In order to complete their life cycle, papillomaviruses have evolved to manipulate a plethora of cellular pathways. The products of the human <i>Alphapapillomavirus</i> E6 proteins specifically interact with and target PDZ containing proteins for degradation. This viral phenotype has been suggested to play a role in viral oncogenesis. To analyze the association of HPV E6 mediated PDZ-protein degradation with cervical oncogenesis, a high-throughput cell culture assay was developed. Degradation of an epitope tagged human MAGI1 isoform was visualized by immunoblot. The correlation between HPV E6-induced degradation of hMAGI1 and epidemiologically determined HPV oncogenicity was evaluated using a Bayesian approach within a phylogenetic context. All tested oncogenic types degraded the PDZ-containing protein hMAGI1d; however, E6 proteins isolated from several related albeit non-oncogenic viral types were equally efficient at degrading hMAGI1. The relationship between both traits (oncogenicity and PDZ degradation potential) is best explained by a model in which the potential to degrade PDZ proteins was acquired prior to the oncogenic phenotype. This analysis provides evidence that the ancestor of both oncogenic and non-oncogenic HPVs acquired the potential to degrade human PDZ-containing proteins. This suggests that HPV E6 directed degradation of PDZ-proteins represents an ancient ecological niche adaptation. Phylogenetic modeling indicates that this phenotype is not specifically correlated with oncogenic risk, but may act as an enabling phenotype. The role of PDZ protein degradation in HPV fitness and oncogenesis needs to be interpreted in the context of <i>Alphapapillomavirus</i> evolution.</p></div

Degradation of PDZ proteins is an enabling phenotype towards oncogenicity.

<p>(A) Table of top five models as selected by the RJMCMC analysis. The RJMCMC was run three independent times to ensure that the reproducibility of the model analysis. Through comparing the ratio of posterior to prior odds (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004980#sec002" target="_blank">materials and methods</a>) we obtain Bayes Factors to support the choice of a specific model. The selected model has a Bayes Factor of 130.41 (st.dev. = 1.99) suggesting decisive evidence in favor of this model. (B) Ancestral phenotype reconstruction at three important nodes of the <i>Alphapapillomavirus</i> phylogeny. The graphs show the estimated marginal probability density plot for the common MRCA, the high-risk MRCA or the low-risk MRCA. The analysis suggests that the MRCA of the high-risk viruses acquired the ability to degrade PDZ-containing protein, but was likely not an oncogenic virus. (C) Estimated instantaneous rates of change between different combinations of viral phenotypes based on the RJMCMC analysis. Histograms show the posterior distribution of estimated values of the rate parameters. The red bars indicate the fraction of samples in which each rate is estimated to be zero. Arrow width is proportional to the median of these estimated rates. Z indicates the percentage of samples in which each rate parameter is estimated as zero. Consistent with the hypothesis that the ability to degrade PDZ containing proteins is an enabling phenotype, rates associated with oncogenic ability independent of PDZ protein interaction are often estimated as zero (i.e. they do not occur), and are thus represented as dotted line.</p

All members of the phylogenetic high-risk clade degrade human MAGI1d.

<p>(A) C-33A cells were transfected with 24 different E6 proteins covering the known evolutionary spectrum within the <i>Alphapapillomavirus</i> genus. The western blot shows a representative experiment. GFP was probed as a transfection control indicating equal transfection. This figure shows that all High-Risk types (highlighted in red) target hMAGI1d for degradation. The pQCXIN vector was used as control. (B) Mirror trees comparing the epidemiological (left) and PDZ-protein degradation (right) phenotypes on the E6 based phylogeny. The viral names are colored according to phylogenetic classification. High-risk viruses are colored in red, while LR viruses are colored green. The branches of the tree are shaded according to the state of each character under investigation.</p

Presence of C-terminal PDZ binding motif is correlated with phylogenetic classification.

<p>The Bayesian phylogenetic tree is constructed based on the E6 nucleotide sequences of all known human <i>Alphapapillomaviruses</i>. HPV8, a <i>Betapapillomaviruses</i> was used to root the tree. The solid branches indicate the optimized branch lengths. Dotted lines were added to the branches to facilitate visual inspection of the tree. Epidemiological classification divides the tree into two main clades (“high-risk” vs.”low-risk”). Oncogenic papillomavirus types are colored red (classification based on [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004980#ppat.1004980.ref007" target="_blank">7</a>]). Posterior probability values are indicated using symbols at the nodes (triangle = 1; rectangle > 0.95; circle>0.90; diamond >0.80). The numbers to the right indicate the different viral species within the genus <i>Alphapapillomaviruses</i>. The sequence of the six C-terminal residues constituting a putative type 1 PBM is indicated following the virus name. A dash (“-“) indicates the absence of a residue at that position. Numbers above the sequences allow for easy identification of landmark residues as in [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004980#ppat.1004980.ref042" target="_blank">42</a>]. Viruses in <b>Bold</b> were selected for the <i>in vivo</i> analyses.</p

HPV E6-mediated p53 degradation.

<p>A) HA-tagged p53 levels were visualized by Western blot after co-transfection with HA-tagged HPV E6 from HPV11, HPV16 and HPV18 into C-33A cells. Lanes 1, 3, 5 and 7 show results from co-transfection of HA-p53 and vectors indicated at the top of the figure. Lanes 2, 4, 6 and 8 show the p53 levels after treatment with MG132, as indicated at the top of the figure. β-tubulin was visualized as a loading control. (B) Half life of HA-p53 in transfected 293T cells. 293T cells were transfected with HA-p53 and control (pQCXIN) or E6 ORFs (HPV11, HPV18, HPV53, HPV56 and HPV66). The band intensities were determined from the scanned Western blot using ImageQuant and the signals at time 0 were defined as 100%. The band intensities of the indicated time points were normalized to time 0.</p

Alignment of HPV alpha E6 ORFs.

<p>The alignment of all 27 E6 ORFs tested in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone-0012816-g002" target="_blank">Figure 2</a> is shown. HPV types degrading p53 are shown at the top of the alignment and those not degrading p53 are shown at the bottom. The shaded region represents a proposed E6-AP binding domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone.0012816-Liu1" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone.0012816-Pim1" target="_blank">[26]</a>. The amino acid sequences of the E6 ORFs were aligned using Clustal X (version 1.81) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone.0012816-Thompson1" target="_blank">[36]</a>. The amino acid at position 31 (arrow, <b>bold</b>) was associated with p53 degradation (p<0.01). “_” indicate gaps, whereas “.” indicates identical residues with the HPV16 E6 amino acid sequence shown in the top row.</p

Phylogeny of alpha-HPVs and degradation of p53 by HPV E6 ORFs.

<p>HPV E6 activity on HA-p53 steady state levels were determined by Western blot using the assay shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone-0012816-g001" target="_blank">Figure 1A</a>. The phylogenetic tree at the left was constructed using the combined early gene sequences as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone.0012816-Narechania1" target="_blank">[35]</a>. Epidemiological carcinogenicity was extrapolated from recent reviews <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone.0012816-Schiffman1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone.0012816-Bouvard1" target="_blank">[24]</a> and is indicated in the column labeled “carcinogenic risk”: ++, highly oncogenic; +, oncogenic; +/−, probably oncogenic; −, not significantly associated with cervix cancer; NA, insufficient data. The p53 levels after co-transfection with E6 from each type indicated on the left are shown in the far right column labeled, “p53” (with and without MG132) and the results are summarized in the column labeled“↓p53”. Endogenous β-tubulin (far right column) represents a loading control. The alpha-HPV species groups are indicated by brackets with a number to the right. The empty vector control, pQCXIN is shown at the bottom.</p

HPV E6 mutagenesis and degradation of p53.

<p>(A) The steady state levels of HA-p53 co-transfected with wild type or mutant HPV71, 90 and 106 HA-E6 in C-33A cells are shown in the row labeled HA-p53. The unmodified E6 ORFs are indicated with “WT” and the mutated E6 ORFs are indicated with the replacement amino acid at the top of the figure. β-tubulin is shown as a loading control in the bottom row. (B) Representative images of double immuno-fluorescence experiments. HA-tagged HPV E6 constructs are green, whereas the endogenous p53 is labeled red. In the p53 image, arrows indicate the location of the E6 expressing cells. Absence of p53 signal (red) is the result of degradation. In the merged images, yellow signifies a non-degrader, whereas green only (HPV E6) is indicative of a p53 degrader. Cell nuclei are detected with DAPI and are shown in the bottom row. Each HPV E6 construct is indicated at the bottom of each column.</p

Midpoint-rooted phylogenetic tree for polyomavirus VP1 protein sequences.

<p>Species with different clade affiliations in LT analyses (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005574#ppat.1005574.g003" target="_blank">Fig 3</a>) are indicated in colored bold oblique text. The script ƒ character indicates fragmentary (sub-genomic) sequences. Percent bootstrap values for selected nodes are indicated. A FigTree file containing detailed bootstrap values is provided as <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005574#ppat.1005574.s008" target="_blank">S3 File</a>. Scale bar shows one substitution per site.</p

A hypothetical framework for ancient recombination events among major polyomavirus clades.

<p>The model attempts to reconcile observed incongruities between LT and VP1 phylogenetic trees shown in Figs <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005574#ppat.1005574.g003" target="_blank">3</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005574#ppat.1005574.g004" target="_blank">4</a>. In the model, a hypothetical ancient polyomavirus, designated Arche, is inferred to have infected the last common ancestor of bilaterian animals. The ancient Arche lineage then gave rise to separate polyomavirus lineages found in arthropods and fish, as well as the mammalian Ortho/Almi lineages. The figure depicts Avi and Wuki clades arising after recombination events involving an unknown vertebrate-Arche lineage and Ortho-like species. The figure does not depict the inferred evolution of the HPyV6/7 clade, which appears to have arisen after a separate recombination event involving the late region of a hypothetical vertebrate-Arche lineage and the early region of a basal Almi-like species. The TSV lineage, which shows evidence of recombination between the Ortho and Almi lineages, is also omitted. White lollipops represent predicted pRb-binding motifs (LXCXE or related sequences). Yellow bars represent hypothetical metal-binding motifs (CXCXXC or related sequences). The absence of metal-binding motifs in Avi small T antigen (sT) proteins suggests a different evolutionary origin than the classic metal-binding Ortho/Almi sT. Possible ALTO-like ORFs predicted for some Ortho species are shaded gray.</p