University of Technology, Sydney. Faculty of Science.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
