Expression and functional characterisation of Variola and Monkeypox virus tumour necrosis factor receptor (TNFR) proteins

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Tumour necrosis factor-a (TNFα) is a pleiotropic cytokine that plays a critical role in cellular response to virus infection. Virtually all poxviruses encode genes that are homologous to human tumour necrosis factor receptors (TNFRs). The viral "T2" TNFR proteins are well characterized from Leporipox viruses, myxoma (Myx) and shape fibroma virus. MyxT2 has previously been shown to bind to and inhibit rabbit TNFα in a species-specific manner and, more recently, has been shown to bind to human cellular TNFRs and inhibit TNFR1-induced cell death in a non species-specific manner. In contrast, the human-tropic Orthopoxviruses TNFR proteins have been poorly characterised, since variola virus (VAR) existed before molecular virology capabilities and the monkeypox virus (MPV) is restricted for research . This study sought to characterize the TNFR proteins, VARG4R and MPVJ2R, encoded by strict species - specific variola virus and the broad host range monkeypox virus, and compare them to the well characterized MyxT2 protein . W ith WHO Smallpox Committee approval, codon optimised cDNAs for VARG4R and MPVJ2R were constructed and these proteins were expressed as C -terminal myc-Histagged fusion proteins by transient transfection in HEK293T cells. Both VARG4R and MPVJ2R are expressed and detectable in cell lysates and culture supernatants, exactly as occurs for MyxT2. However, while MyxT2 is both a dimer and a monomer, VARG4R is predominantly a dimer and MPVJ 2R is exclusively a monomer. Secreted VARG4R and MPVJ2R are heavily glycosylated consistent with their numerous N-linked glycosylation sites. In TNFα neutralization L929 cytotoxicity assays, VARG4R inhibits rabbit, mouse, human and rhesus macaque TNFα. Although monkeypox virus has an extremely broad host range, surprisingly MPVJ2R has no TNFα inhibitory activity against rabbit, human, or rhesus macaque TNFα. Consistent with MyxT2, VARG4R and MPVJ2R inhibit TNFR1-induced cell death. It has previously been demonstrated that the viral pre-ligand association domain (PLAD) is essential for MyxT2 inhibition of TNFRl-induced cell death. Interestingly, MyxT2 PLAD, VARG4R PLAD and MPVJ2R PLAD proteins also inhibit TNFRl-induced cell death, confirming the critical role of the viral PLAD domain in the function of these viral TN FR proteins. Collectively these data suggest that, like MyxT2, secreted VARG4R protein acts as a functional TNFα-inhibitory factor during variola infection, but poxviruses with a broad host range, such as monkeypox, use other non species-specific mechanisms for host immune evasion . Overall, this study expands on our limited knowledge of variola and monkeypox viruses' mechanisms of immune evasion and further confirms the pivotal role of the viral PLAD in viral inhibition of TNFα-TN FR signaling.
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