A chemokine-binding domain in the tumor necrosis factor receptor from variola (smallpox) virus

Abstract

Variola virus (VaV) is the causative agent of smallpox, one of the most devastating diseases encountered by man, that was eradicated in 1980. The deliberate release of VaV would have catastrophic consequences on global public health. However, the mechanisms that contribute to smallpox pathogenesis are poorly understood at the molecular level. The ability of viruses to evade the host defense mechanisms is an important determinant of viral pathogenesis. Here we show that the tumor necrosis factor receptor (TNFR) homologue CrmB encoded by VaV functions not only as a soluble decoy TNFR but also as a highly specific binding protein for several chemokines that mediate recruitment of immune cells to mucosal surfaces and the skin, sites of virus entry and viral replication at late stages of smallpox. CrmB binds chemokines through its C-terminal domain, which is unrelated to TNFRs, was named smallpox virus-encoded chemokine receptor (SECRET) domain and uncovers a family of poxvirus chemokine inhibitors. An active SECRET domain was found in another viral TNFR (CrmD) and three secreted proteins encoded by orthopoxviruses. These findings identify a previously undescribed chemokine-binding and inhibitory domain unrelated to host chemokine receptors and a mechanism of immune modulation in VaV that may influence smallpox pathogenesis.immune evasion ͉ viral pathogenesis ͉ cytokine receptor ͉ poxvirus ͉ inflammation

Comparative Biochemical and Functional Analysis of Viral and Human Secreted Tumor Necrosis Factor (TNF) Decoy Receptors

“…Genes were PCR-amplified from recombinant plasmids, except for CrmD from CPXV strain Brighton Red (CPXV221; GenBank TM accession number AAM13659), which was amplified from viral genomic DNA. CrmB (CPXV005; GenBank TM AAA60952) and CrmC (CPXV191; GenBank TM AAM13631) from CPXV strain Brighton Red were PCR-amplified from pAH21 and pRA112 (22), respectively. CrmE (K3R; GenBank TM AJ272008) from CPXV strain Elephantpox was amplified from pMS3 (13).…”

“…Permission from the World Health Organization was granted to hold VARV DNA, and its manipulation was performed in accordance to the established rules. A plasmid containing this sequence, pRA107, was used as template (22). pMS1 (13) was used to amplify the CrmD gene (E6; GenBank TM AJ567688) from ECTV strain Hampstead.…”

“…Recombinant baculoviruses were generated using the Bacto-Bac system (Life Technologies) (22). Viral stocks were amplified by infecting Hi5 cells at low viral multiplicity of infection (0.1-0.01 pfu/cell) to obtain high titer baculovirus stocks for protein production.…”

“…Thus, variola virus (VARV), the causative agent of smallpox (21), encodes one copy of a single active vTNFR, CrmB that can efficiently inhibit human TNF and LT␣ (22). CrmB is also the only active vTNFR gene in monkeypox virus (MPXV) that harbors two copies of this vTNFR.…”

Poxvirus-encoded TNF decoy receptors inhibit the biological activity of transmembrane TNF

“…In addition, we recently identified LTb as a new ligand for CrmD and CrmB (Pontejo et al, 2015). Importantly, CrmB and CrmD, but not CrmC and CrmE, contain an extended Cterminal chemokine-binding domain termed SECRET (Alejo et al, 2006). Previous data support a role of vTNFRs in poxvirus pathogenesis.…”