Interactions of viral and cellular Tumour Necrosis Factor Receptor molecules

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Tumour necrosis factor (TNF) is potent pro-inflammatory and anti-viral cytokine, acting via two cellular receptors, TNFR1 and TNFR2 that induces apoptosis and inflammation. Poxviruses encode homologues of TNF-receptors (viral TNFRs) that independently interact with both TNF, and simultaneously with cellular TNFRs, to subvert TNF-induced anti-viral apoptosis. The vTNFRs are expressed during poxvirus infection and are considered as bona fide virulence factors. The recently discovery of a “Pre-ligand Assembly Domain (PLAD)” within the N-terminus of the cellular TNFRs is shown to be required for receptor trimerisation and efficient cell death signalling. Whilst it has previously shown that the rabbit-trophic Myxoma (MYX) viral TNFR also contains a PLAD required for viral TNFR:cellular TNFR interactions, little is known about the human-trophic poxvirus TNFRs, nor physical characteristics of the interactions of vTNFRs and cellular TNFRs. To assess the importance of the PLAD domain in TNFR structure, function and viral subversion of TNFRs, this study focused on naturally occurring mutations in the TNFR PLAD domain, that occur in transient periodic fevers (TRAPS) – a clinical syndrome of febrile attacks of inflammation. TRAPS PLAD domain mutations were generated in a TNFR1-YFP in plasmids by site-directed mutagenesis and cloning. WT and TRAPS mutant TNFR1 constructs were transfected into U20S cells and TNFR1 location was determined by confocal microscopy. Neither WT TNFR1 nor TRAPS TNFRs were unable to be detected at the cell surface by both widefield and confocal microscopy despite published data on surface expression of WT TNFR1. WT TNFR1-YFP fusion proteins were found to be expressed within endocytic vesicles known as receptosomes and also as aggregates in a membranous structure resembling Golgi/ER. In addition it was found that TRAPS mutations in particular those affecting critical amino acids such as cysteines in disulphide bonds, display reduced TNFR-induced cell death as determined by flow cytometry. To better understand the biology of the vTNFR association with cellular TNFRs, and with WHO Smallpox committee approval, the human tropic poxviral TNFRs from Variola (Smallpox) (VAR) and Monkeypox (MPV) were synthesised and cloned as CFP/YFP and MycHis expression plasmids. Using multi-colour flow cytometry we have shown that, like the MYXT2 vTNFR, VARG4R and MPVJ2R TNFRs are potent intracellular inhibitors of TNFR1-induced cell death. As each vTNFR was able to inhibit TNFR-induced cell death, an assay was developed by flow cytometry to measure the intracellular abundance of the vTNFRs in the presence of cellular TNFR overexpression. MYXT2 was found to increase in intracellular abundance however for unknown reasons VARG4R and MPVJ2R did not convincingly increase in abundance. A structure for each of the vTNFRs was then attempted to be determined by X-ray crystallography, however bacterial expression of the both the cellular TNFRs and viral TNFRs proteins were unable to be obtained. Lastly to determine the structural orientations and conformations of cellular vTNFR interactions, a method of fluorescence resonance energy transfer (FRET) was established by flow cytometry. Using the generated C-terminal fusion -CFP and -YFP TNFRs, interactions were assessed between each of the cellular and vTNFRs. It was found that in addition to the reduced cell death TRAPS TNFRs when expressed with WT TNFR1, TRAPS mutations also cause reduced FRET possibly due to altered conformations in the receptor. Again mutations affecting more critical structural amino acids were found to have a more dramatic effect. Moreover differences were observed between mutations in distribution of FRET histograms further indicating altered network formations of higher order complexes. Next the FRET method was used to assess interactions between each of the vTNFRs with WT human TNFRs as well as with themselves and other vTNFRs. However no FRET was detectable between each of the molecules despite evidence of MYXT2 associating with human TNFR1 and TNFR2. Thus Comparative homology modelling and automated docking simulations were performed to explain possible orientations of the interactions tested in FRET. These data suggest that the interactions of vTNFRs with cellular TNFRs may possibly occur in a C-N anti-parallel orientation and not the previously predicted PLAD-PLAD interactions. Taken together, these data further our understanding of basic TNFR biology as well as for the first time characterise an entire panel of PLAD TRAPS mutations. It also furthers the characterisation of the very limited evidence of vTNFR subversion of TNFRs for the human trophic viral proteins VARG4R and MPVJ2R. Overall these results show the importance of PLAD interactions to TNFR biology and a possible new avenue in which TNFR signalling may be exploited in the development of new therapeutics.
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