Bovine papillomavirus type 2 (BPV-2) E5 oncoprotein binds to the subunit D of the V₁-ATPase proton pump in naturally occurring urothelial tumors of the urinary bladder of cattle

Abstract

Background: Active infection by bovine papillomavirus type 2 (BPV-2) was documented for fifteen urinary bladder tumors in cattle. Two were diagnosed as papillary urothelial neoplasm of low malignant potential (PUNLMP), nine as papillary and four as invasive urothelial cancers.

Methods and findings: In all cancer samples, PCR analysis revealed a BPV-2-specific 503 bp DNA fragment. E5 protein, the major oncoprotein of the virus, was shown both by immunoprecipitation and immunohistochemical analysis. E5 was found to bind to the activated (phosphorylated) form of the platelet derived growth factor β receptor. PDGFβR immunoprecipitation from bladder tumor samples and from normal bladder tissue used as control revealed a protein band which was present in the pull-down from bladder cancer samples only. The protein was identified with mass spectrometry as "V₁-ATPase subunit D", a component of the central stalk of the V₁-ATPase vacuolar pump. The subunit D was confirmed in this complex by coimmunoprecipitation investigations and it was found to colocalize with the receptor. The subunit D was also shown to be overexpressed by Western blot, RT-PCR and immunofluorescence analyses. Immunoprecipitation and immunofluorescence also revealed that E5 oncoprotein was bound to the subunit D.

Conclusion: For the first time, a tri-component complex composed of E5/PDGFβR/subunit D has been documented in vivo. Previous in vitro studies have shown that the BPV-2 E5 oncoprotein binds to the proteolipid c ring of the V₀-ATPase sector. We suggest that the E5/PDGFβR/subunit D complex may perturb proteostasis, organelle and cytosol homeostasis, which can result in altered protein degradation and in autophagic responses.

Figure 1. PCR amplification and sequencing of BPV-2 DNA.

Lane M, Molecular mass marker (1 kb DNA Ladder, Microtech). Lanes 1–3: Three normal (control) samples from healthy cows. Lanes 4–6: three representative tumor samples. Lane C+: positive control containing a cloned BPV-2 DNA. Lane C-. negative control (no DNA added). The lower part of the figure shows 100% identity between the sequence of the amplicons in lanes 4–6 and the sequence of BPV-2 deposited in GenBank (M20219.1).

Figure 2. BPV-2 E5 immunoprecipitation.

The presence of E5 protein detected by immunoprecipitation. a) Lanes 1–3: urinary bladders from healthy cows. Lanes 4–6: three representative urothelial tumors of the urinary bladder in cows. Lanes 7: positive control (bovine placenta infected with BPV-2).

Figure 3. BPV-2 E5 immunohistochemistry.

Urothelial carcinoma. Immunohistochemical detection of cytoplasmic E5 in neoplastic urothelial cells. E5-expressing cancer cells are scattered both in basal and suprabasal layers. Magnification, ×550. Insert: normal (control) urothelium from healthy cows. Magnification, ×550.

Figure 4. Total and phosphorylated (activated) PDGFβR expression.

(x) Total protein extracts from tissue lysates were generated and used in Western blot analysis with an antibody specific for total PDGFβR and a phosphospecific PDGFβR antibody that recognized pPDGFβR phosphorylated at Tyr770. Lanes 1–3: urinary bladder from healthy animals. Lanes 4–6: representative neoplastic tissues from three cows with papillomavirus-associated tumors of the urinary bladder. Actin protein levels were detected to ensure equal protein loading. (y) Quantitative densitometric analysis of the filters was performed with Image Lab software (ChemiDoc; Bio-Rad Laboratories) and significance determined by the Student T-test (***, p<0.001).

Figure 5. pPDGFβR immunohistochemistry.

Urothelial carcinoma. Immunohistochemical detection of phosphorylated PDGFβR in neoplastic urothelial cells and in normal (control) urothelial cells as shown in the insert. Magnification, ×550.

Figure 6. BPV-2 E5 and PDGFßR co-immunoprecipitation.

The presence of phosphorylated and total PDGFßR was detected in E5 immunoprecipitates. Lanes 1–3: urinary bladder from healthy cows. Lanes 4–6: cancer tissue from three cows with papillomavirus-associated tumors of the urinary bladder.

Figure 7. E5 and pPDGFβR colocalization.

Urothelial carcinoma. Immunofluorescence detection of E5 and pPDGFβR and their colocalization (yellow in the merged images) in urothelial cancer cells (upper) vs normal (control) urothelial cells (lower). Magnification ×550.

Figure 8. PDGFßR and V₁-ATPase co-immunoprecipitation.

PDGFβR interaction with V₁-ATPase subunit D is increased in immunoprecipitates derived from pathological tissues. V₀-ATPase c subunit does not co-immunoprecipitate with PDGFβR. Lanes 1–3: urinary bladder from healthy cows. Lanes 4–6: cancer tissue from three cows with papillomavirus-associated tumors of the urinary bladder.

Figure 9. V₁-ATPase subunit D and pPDGFβR colocalization.

Urothelial carcinoma. Immunofluorescence detection of the V₁-ATPase subunit D and the pPDGFβR and their colocalization signal (yellow in the merged image). Magnification, ×550.

Figure 10. V₁-ATPase subunit D expression.

(x) Western blot analysis showing overexpression of V₁-ATPase subunit D. Lanes 1–3: urinary bladder from healthy animals. Lanes 4–6: neoplastic tissue from representative three cows with papillomavirus-associated tumors of the urinary bladder. Actin protein levels were detected to ensure equal protein loading. (y) Quantitative densitometric analysis of the filters was performed with Image Lab software (ChemiDoc; Bio-Rad Laboratories) and significance determined by the Student T-test (**, p<0.01).

Figure 11. V₁-ATPase subunit D immunofluorescence.

Urothelial carcinoma. The overexpression of the subunit D of the V₁-domain is also detected by immunofluorescence. The subunit D appears to be overexpressed both in the membrane and in the cytoplasm of urothelial cancer cells compared to urothelial normal cells (insert). Magnification, ×550.

Figure 12. RT PCR for subunit D.

The relative expression levels of V₁-ATPase subunit D in neoplastic tissues. V₁-ATPase subunit D mRNA levels were determined by qRT-PCR. Relative mRNA levels, calculated using the ΔΔC_T method, represent fold changes in comparison to urinary bladder samples from healthy cows. All values were normalized to the internal control β-actin. Results represent the means and standard deviations of three independent experiments performed in triplicate. (*, p<0.05, vs urinary bladder from healthy cows).

Figure 13. V₀-ATPase c subunit expression.

Western blot analysis showing similar expression levels of V₀-ATPase c subunit among all the samples. Lanes 1–3: urinary bladder from healthy animals. Lanes 4–6: neoplastic tissue from representative three cows with papillomavirus-associated tumors of the urinary bladder. Actin protein levels were detected to ensure equal protein loading.

Figure 14. BPV-2 E5 and V₁-ATPase subunit D co-immunoprecipitation.

The presence of V₁-ATPase subunit D was detected in E5 immunoprecipitates. Lanes 1–3: urinary bladder from healthy cows. Lanes 4–6: neoplastic tissue from three cows with papillomavirus-associated tumors of the urinary bladder.

Figure 15. V₁-ATPase subunit D and BPV-2 E5 colocalization.

Urothelial carcinoma. Immunofluorescence detection of E5 and the subunit D. The proteins appear to colocalize (yellow in the merged image).

Figure 16. BPV-2 E5, PDGFßR and V₁-ATPase D subunit co-immunoprecipitation.

The presence of PDGFßR and V₁-ATPase D subunit was detected in E5 immunoprecipitates. Lanes 1–3: urinary bladder from healthy cows. Lanes 4–6: tissue from three cows with papillomavirus-associated tumors of the urinary bladder.