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Structural role of the conserved cysteines in the dimerization of the viral transmembrane oncoprotein E5.

ABSTRACT: The E5 oncoprotein is the major transforming protein of bovine papillomavirus type 1. This 44-residue transmembrane protein can interact with the platelet-derived growth factor receptor ?, leading to ligand-independent activation and cell transformation. For productive interaction, E5 needs to dimerize via a C-terminal pair of cysteines, though a recent study suggested that its truncated transmembrane segment can dimerize on its own. To analyze the structure of the full protein in a membrane environment and elucidate the role of the Cys-Ser-Cys motif, we produced recombinantly the wild-type protein and four cysteine mutants. Comparison by circular dichroism in detergent micelles and lipid vesicular dispersion and by NMR in trifluoroethanol demonstrates that the absence of one or both cysteines does not influence the highly ?-helical secondary structure, nor does it impair the ability of E5 to dimerize, observations that are further supported by sodium dodecylsulfate polyacrylamide gel electrophoresis. We also observed assemblies of higher order. Oriented circular dichroism in lipid bilayers shows that E5 is aligned as a transmembrane helix with a slight tilt angle, and that this membrane alignment is also independent of any cysteines. We conclude that the Cys-containing motif represents a disordered region of the protein that serves as an extra covalent connection for stabilization.

SUBMITTER: Windisch D

PROVIDER: S-EPMC2941001 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Structural role of the conserved cysteines in the dimerization of the viral transmembrane oncoprotein E5.

Windisch Dirk D Hoffmann Silke S Afonin Sergii S Vollmer Stefanie S Benamira Soraya S Langer Birgid B Bürck Jochen J Muhle-Goll Claudia C Ulrich Anne S AS

Biophysical journal 20100901 6

The E5 oncoprotein is the major transforming protein of bovine papillomavirus type 1. This 44-residue transmembrane protein can interact with the platelet-derived growth factor receptor β, leading to ligand-independent activation and cell transformation. For productive interaction, E5 needs to dimerize via a C-terminal pair of cysteines, though a recent study suggested that its truncated transmembrane segment can dimerize on its own. To analyze the structure of the full protein in a membrane env ...[more]

PMID: 20858420

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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.

Dataset Information

Structural role of the conserved cysteines in the dimerization of the viral transmembrane oncoprotein E5.

Publications

Structural role of the conserved cysteines in the dimerization of the viral transmembrane oncoprotein E5.

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