Dataset Information

Bovine papillomavirus type 2 (BPV-2) E5 oncoprotein binds to the subunit D of the V?-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 ? receptor. PDGF?R 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 "V?-ATPase subunit D", a component of the central stalk of the V?-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/PDGF?R/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 V?-ATPase sector. We suggest that the E5/PDGF?R/subunit D complex may perturb proteostasis, organelle and cytosol homeostasis, which can result in altered protein degradation and in autophagic responses.

SUBMITTER: Roperto S

PROVIDER: S-EPMC3933332 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Publications

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.

Roperto Sante S Russo Valeria V Borzacchiello Giuseppe G Urraro Chiara C Lucà Roberta R Esposito Iolanda I Riccardi Marita Georgia MG Raso Cinzia C Gaspari Marco M Ceccarelli Dora Maria DM Galasso Rocco R Roperto Franco F

PloS one 20140224 2

<h4>Background</h4>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.<h4>Methods and findings</h4>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 immunohistoche ...[more]

PMID: 24586417

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Project description:Persistent infection and tumorigenesis by papillomaviruses (PVs) require viral manipulation of various cellular processes, including those involved in innate immune responses. The cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes (cGAS-STING) pathway has emerged as an essential innate immune sensing system, that recognizes DNA and trigger potent antiviral effector responses. In this study, we found that bovine PV (BPV) E5 protein, the major oncoprotein of bovine delta PVs, interacts with STING but not with cGAS in a spontaneous BPV infection of neoplastic urothelial cells of cattle. Real-time RT-PCR revealed a significant reduction in both cGAS and STING transcripts in E5-expressing cells. Furthermore, western blot (WB) analysis failed to detect any variation in the expression of interferon-inducible protein 16 (IFI16), an upstream effector of the STING pathway. A ternary complex composed of E5/STING/IFI16 was also observed. Co-immunoprecipitation studies showed that STING interacts with a protein network composed of total and phosphorylated TANK-binding kinase 1 (TBK1), total and phosphorylated interferon regulatory factor 3 (IRF3), IRF7, IKKα, IKKβ, IKKϵ, ELKS, MEKK3, and TAK1. RT-qPCR revealed a significant reduction in TBK1 mRNA levels in BPV-infected cells. WB analysis revealed significantly reduced expression levels of pTBK1, which is essential for the activation and phosphorylation of IRF3, a prerequisite for the latter to enter the nucleus to activate type 1 IFN genes. WB also revealed significantly down-expression of IKKα, IKKβ, IKKϵ, and overexpression of IRF7, ELKS, MEKK3, and TAK1in BPV-positive urothelial cells compared with that in uninfected healthy cells. Phosphorylated p65 (p-p65) was significantly reduced in both the nuclear and cytosolic compartments of BPV-infected cells compared with that in uninfected urothelial cells. Our results suggest that the innate immune signaling pathway mediated by cGAS-STING is impaired in cells infected with BPV. Therefore, effective immune responses are not elicited against these viruses, which facilitates persistent viral infection and subsequent tumorigenesis.