Molecular analyses of tumor-bearing animals.

<p><b>A)</b> Viral load in tissue samples from UV-irradiated and control animals from the MnPV-infected colony analyzed by qPCR and normalized to a plasmid standard. Samples were grouped according to their origin as indicated (ctrl skin: skin from unirradiated animals; ui skin/UV skin: unirradiated or UV-irradiated skin from irradiated animals; KSCC/nKSCC: UV-induced SCCs; non-UV tumor: tumors from non-UV sites of irradiated animals and spontaneous tumors from unirradiated animals). UV^+/- indicates whether the animal was UV-exposed or not (Kruskal-Wallis test, *p<0.05, ***p<0.001, ^nsp>0.05). <b>B)</b> Southern blot analysis of unirradiated and UV-irradiated skins, a KSCC and a non-UV tumor. DNA was digested with ApaI (no cleavage site in MnPV), XbaI (one site) or XhoI (two sites) as indicated (Form I: supercoiled; Form II: relaxed circular; Form III: linear form of MnPV). <b>C)</b> Semi-quantitative RT-PCR for the most abundant MnPV <i>E1^E4</i> transcript in non-UV tumors and UV-induced SCCs or the control <i>GAPDH</i>. <b>D)</b> Semi-quantitative RT-PCR for MnPV <i>E6</i>, <i>E7</i> and <i>L1</i> transcripts in non-UV tumors and UV-induced SCCs or the control <i>GAPDH</i>.</p

Transactivating capacity of <i>Mastomys</i> p53 in the presence of MnPV E6.

<p><b>A)</b> The capacity of p53 to transactivate a p53-responsive firefly luciferase gene measured in H1299 cells transfected with reporter plasmids and expression vectors for <i>Mastomys</i> p53 and MnPV E6 or human p53 and HPV16 E6 as a control. Transactivation activity was measured by luminescence (RLU, relative light units). Cells transfected only with p53 served as control and their RLU levels were arbitrarily set to 1 (Mean ± SEM; n = 7; 1way-ANOVA, ***p<0.0001). <b>B)</b> Western blots showing protein levels of p53 and E6 in the lysates of the transactivation assay. Actin served as an internal loading control.</p

MnPV interferes with DNA damage repair.

<p><b>A)</b> Repair kinetics of CPDs in MnPV E6/E7-positive and -negative <i>Mastomys</i> keratinocytes (Mean ± SD; n = 2, measurements were performed in quadruplicates). <b>B)</b> Immunofluorescence staining of γH2AX foci in keratinocytes stably expressing MnPV E6/E7. Cells were irradiated with UVB and further incubated prior to detection and quantification of γH2AX foci (Ctrl: unirradiated, UV: irradiated; Red: γH2AX, blue: nuclei; scale bars: 50 μm). <b>C)</b> Quantification of γH2AX foci (Mean ± SEM; n≥242; 1way-ANOVA, *p<0.05, **p<0.01, ***p<0.001). <b>D)</b> Co-detection of CPDs and γH2AX in MnPV^+/- skin harvested 24h after UV irradiation. Arrows point towards positive cells (Viral loads: animal 3: 13.68 ± 1.66 copies/cell, animal 4: 147.42 ± 14.62 copies/cell; Scale bars: 100 μm). <b>E)</b> Co-detection of CPDs and γH2AX in a KSCC harvested 24h after UV irradiation (Viral load: 611.88 ± 18.75 copies/cell; scale bars: 100 μm).</p

Summary of the absolute numbers of tumors in the different groups, percentage of tumor-bearing animals and median time of tumor development.

<p>Summary of the absolute numbers of tumors in the different groups, percentage of tumor-bearing animals and median time of tumor development.</p

Dedifferentiation correlates with positive p53 staining.

<p>Consecutive sections of a poorly differentiated nKSCC were stained with antibodies against E-cadherin, vimentin, pan-Cytokeratin and p53. DAPI was used as nuclear counter stain. Note that in this tumor, only mutation R266C could be detected (Scale bars: 100 μm).</p

Histological analyses of a non-UV tumor and UV-induced tumors.

<p><b>A)</b> Tumors from unirradiated sites show papilloma-like growth of well-differentiated neoplastic squamous cells (H&E). Especially the basal layers are hyperproliferative as indicated by strong Ki-67 staining. Throughout all layers of the lesion, neoplastic cells strongly express cytokeratins (pan-Cytokeratin). <b>B)</b> A UV-induced KSCC with well-differentiated exoendophytic proliferations of squamous cells expressing Ki-67 throughout all neoplastic squamous layers. <b>C and D)</b> In some cases well-differentiated KSCCs <b>(C)</b> further developed into more aggressive poorly differentiated nKSCCs <b>(D)</b>. Proliferating altered squamous cells thereby invaded deeper layers and often changed to a spindle-like phenotype (H&E). The Ki-67 staining becomes diffuse in this process (compare insets) and cytokeratin expression is reduced. (d: dermis; e: epidermis; f: fat; k: keratin; m: muscle; u: ulceration; t: tumor. Scale bars: macroscopic: 10 mm, overviews: 1 mm, insets 100 μm).</p

Study design and tumor development.

<p><b>A)</b><i>Mastomys coucha</i> as a model for cutaneous papillomavirus infection. In the study, naturally MnPV-infected animals (MnPV⁺) as well as virus-free control animals (MnPV^-) were irradiated three times per week with UVB. The starting dose of 150 mJ/cm² was increased weekly by 50 mJ/cm² until the desired final dose was reached (450, 600 or 800 mJ/cm², respectively). Black arrows indicate an increase of the dose, gray arrows the subsequent application of this dose. The irradiation was continued until the animals were sacrificed or died. <b>B)</b> Kaplan-Meier curves demonstrating the percentage of irradiated virus-infected (MnPV⁺, UV⁺), virus-free (MnPV^-, UV⁺) and unirradiated virus-infected (MnPV⁺, UV^-) tumor-bearing animals. <b>C)</b> Two examples of spontaneous skin lesions arising in naturally infected animals. <b>D)</b> Examples of UV-induced keratinizing SCCs (KSCC) with similarities to human keratoacanthomas. <b>E)</b> Examples of UV-induced non-keratinizing SCCs (nKSCC) (C, D and E: scale bars: 10 mm). <b>F)</b> Number of KSCCs and nKSCCs in correlation with the final UV doses. Note that KSCCs preferentially appeared at the lowest dose, nKSCCs preferentially at higher doses (Mean ± SEM; animal numbers: see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006723#ppat.1006723.t001" target="_blank">Table 1</a>; av: average number of tumors).</p

Schematic overview of the mechanism suggested for UV-induced NMSC development in <i>Mastomys coucha</i>.

<p><b>A)</b> MnPV infects basal epithelial cells of the skin of young animals via small injuries. <b>B)</b> MnPV genome is amplified in stratified skin layers (pink and red nuclei) and new virions are released. <b>C)</b> UVB irradiation of the skin. <b>D)</b> UVB-irradiated skin is hyperproliferative, favoring viral replication and virion formation. UVB-induced photoproducts, e.g. in <i>Trp53</i>, occur in keratinocytes (altered nuclei). In uninfected cells, damages are repaired. In infected cells, MnPV-E6/E7 reduce chromosomal stability and inhibit DNA repair. Mutations can accumulate and altered cells become neoplastic. <b>E)</b> Neoplastic squamous cells (light blue) start forming a well-differentiated keratinizing SCC, still representing a permissive system that allows viral replication and formation of virions. <b>F)</b> When neoplastic squamous cells accumulate further mutations (dark blue), a spindle cell phenotype is acquired, forming a poorly differentiated SCC that may become ulcerated. MnPV cannot replicate in dedifferentiated cells and the viral DNA is subsequently lost.</p

Collagen IV staining on tissue sections reveals invasion of keratinocytes through the basal membrane (BM).

<p>The BM was stained against collagen IV (green). Nuclei were counterstained with DAPI (blue). Consecutive sections stained for pan-cytokeratin are shown in comparison. <b>A)</b> In normal skin, the BM (white arrows) marks the barrier between epidermis and dermis. <b>B)</b> Early stage carcinoma formation in UV-irradiated skin. A lack of collagen IV expression indicates the disruption of the BM (orange arrows) accompanied by downward migrating cells (black arrow). <b>C)</b> In the edge region of a UV-induced KSCC, the BM is lost and invading altered keratinocytes are detectable. <b>D)</b> In nKSCC, invasion of neoplastic cells is advanced as indicated by pan-cytokeratin staining. The discontinuous staining of the BM marks transition zones where invading neoplastic squamous cells acquire a spindle cell phenotype (Scale bars: 100 μm).</p

Spatial analysis of viral load in UV-induced SCCs.

<p>Quantification and distribution of MnPV DNA in different microdissected areas (specified in the HE staining) of <b>A)</b> a KSCC and <b>B)</b> a UV-induced nKSCC. Asterisks indicate the position of the respective MnPV-specific <i>in situ</i> hybridization (ISH) (Scale bars: HE: 1 mm, ISH: 100 μm).</p