Tumour
necrosis factor (TNF) is potent pro-inflammatory and anti-viral cytokine, which
interacts with two cellular receptors TNFR1 and TNFR2 (cTNFRs) to induce apoptosis
and inflammation. Poxviruses encode homologues of TNFR (vTNFRs) that
independently interact with TNF and simultaneously with cTNFRs, to inhibit TNF/TNFR-induced
apoptosis. With WHO Smallpox committee approval, we have codon optimised,
expressed, and characterised the human tropic Orthopoxviral TNFRs,
Variola/Smallpox (VARG4R) and monkeypox (MPVJ2R), as well as the Myxoma
Leporipox virus (MYXT2) proteins. Here, we show that Orthopoxvirus vTNFRs are
present in intracellular compartments as well as in secreted forms; intracellular
TNFRs co-localise with cTNFR in Rab-5 positive early endosomes and the Orthopox
vTNFRs are also potent intracellular inhibitors of TNFR1-induced cell death.
To
better understand the significance of these interactions we next examined the
interactions of vTNFRs with naturally occurring cTNFR mutations that are
causative of inherited periodic fevers – TNF-Associated Periodic Syndrome
(TRAPS). Given that there is no known biological “benefit” for TRAPS mutations,
we hypothesized that TRAPs TNFR proteins might no longer associate with viral
TNFRs, and hence that the TRAPS mutations might act to mitigate against the
viral TNFRs ability to inhibit TNFR-induced apoptosis. Here, we present
experiments using YFP-, CFP- and Myc-epitope-tagged TNFRs, in a novel
fluorescence resonance energy transfer (FRET)-based flow cytometry assay to examine
the interactions of vTNFRs with WT and TRAPS mutant TNFRs. Understanding the
nature of TNFR molecular interactions reveals how poxviruses inhibit TNFR1-induced
cell death whilst simultaneously informing the development of the next
generation of anti-inflammatory, TNFR-specific, therapeutic drugs.