In the absence of CrmD, an increased inflammatory infiltrate was observed both in the footpad and in the liver, consistent with the chemokine inhibitory function of its SECRET domain

In the absence of CrmD, an increased inflammatory infiltrate was observed both in the footpad and in the liver, consistent with the chemokine inhibitory function of its SECRET domain. of the smallpox-like disease mousepox. Here we show that CrmD is an essential virulence factor that controls natural killer cell activation and allows progression of fatal mousepox, and demonstrate that both SECRET and TNF binding domains are RG3039 required for full CrmD activity. Vaccination with recombinant CrmD protects animals from lethal mousepox. These results indicate that a specific set of chemokines enhance the inflammatory and protective anti-viral responses mediated by TNF and lymphotoxin, and illustrate how viruses optimize anti-TNF strategies with the addition of a chemokine binding domain as soluble decoy receptors. Introduction A unique immune evasion mechanism employed by poxviruses and herpesviruses is the production of soluble binding proteins or secreted versions of host receptors that neutralize cytokines1C4. The poxvirus homologs of host tumour necrosis factor TNF (TNF) receptors (vTNFRs) block the pro-inflammatory activity of some TNF superfamily (TNFSF) ligands. Five vTNFRs have been described in poxviruses, a viral homolog of host TNFSF receptor CD30 and four TNF inhibitors named cytokine response modifiers B, C, D, and E (CrmB, CrmC, CrmD, and CrmE). vTNFRs are differently expressed among viral species and show distinct binding and inhibitory properties5C13. While CrmE and CrmC are specific TNF inhibitors, CrmD and CrmB block TNF and lymphotoxin (LT). Furthermore, we have recently described that vTNFRs can inhibit membrane TNF and that CrmD and CrmB neutralize another TNFSF ligand, LT14,15. In addition to the cysteine-rich domains (CRDs), characteristic of the ligand binding region of cellular TNFRs, CrmB and CrmD have a C-terminal domain unrelated to host proteins that binds chemokines and was named SECRET (smallpox virus-encoded chemokine receptor) domain16. The crystal structure of the CrmD SECRET domain showed a beta-sandwich fold related to that of the viral chemokine binding proteins (vCKBPs) 35-kDa and A41, but a different chemokine connection region may confer its unique narrower binding specificity17C19. Such variety of activities may provide poxviruses the ability to differentially block chemokines involved in unique anti-viral reactions, to inhibit chemokines at different phases of illness in the sponsor or to simultaneously inhibit chemokines and TNF. Interestingly, the beta-sandwich collapse of vCKBPs has also been observed in additional unrelated poxviral proteins including CPXV203, a major histocompatibility complex I binding protein encoded by cowpox disease (CPXV), and GIF, the granulocyte-macrophage colony-stimulating element and interleukin 2 inhibitor of the parapox Orf disease4,20. To reflect such diverse range of immunomodulatory activities this folding has been named as poxvirus immune evasion domain4,20. ECTV is definitely a mouse-specific orthopoxvirus21,22 genetically related to vaccinia disease (VACV), variola disease (VARV) (the causative agent of human being smallpox) and monkeypox disease (MPXV)23,24, a human being pathogen whose incidence is increasing due to the cessation of mass smallpox vaccination in Africa25,26. Vulnerable strains of mice infected with ECTV develop mousepox, a severe disease that constitutes a good model for smallpox. ECTV RG3039 illness of vulnerable mouse strains via the s.c. route has been exploited like a model of generalized disease infections, genetic resistance to disease, and viral immunology21,22,27. In ECTV, CrmD is the only secreted TNFR. Similarly, both VARV and MPXV communicate a single vTNFR with related characteristics, CrmB. By contrast, CPXV expresses four unique vTNFRs13. In addition, ECTV and additional poxviruses encode RG3039 intracellular proteins that inhibit TNF-induced signalling, underscoring the importance of TNF in antiviral reponses18,28. However, our knowledge of the part of TNF and LT in the control of poxviral infections in vivo is limited. Knockout mice lacking both TNFR1 and TNFR2 showed a slightly improved susceptibility to ECTV and elevated viral replication, with 60% of the infected animals succumbing to mousepox while all WT mice resisted illness29. A direct antiviral activity of TNF has been proposed using a recombinant VACV expressing TNF30. This direct effect was substantiated in TNF-deficient mice infected with VACV, which showed a moderate (two-fold) reduction in LD50 as compared to WT mice, that was accompanied by an increased disease load but not by a diminished T cell response31. Although resistance to mousepox was associated with Th1-like cytokine manifestation, including TNF, blockade of TNF using monoclonal antibodies did not impact the generation of NK cell and CTL reactions, disease clearance or resistance to ECTV illness32 and treatment with TNF did only reduce the mortality rate from 100% to 70% in vulnerable BALB/c mice33. VACV-infected TNFR2-deficient C57BL6 mice produced higher viral titers in spleens and livers and reduced numbers of inflammatory cell foci in the liver, as compared to SMN WT mice34. A contribution of vTNFRs to pathogenesis was initially demonstrated having a CPXV lacking CrmB, but expressing additional vTNFRs, which displayed an increased LD50 in infected mice after intracranial inoculation, a route of infection not natural for poxviruses35. Inactivation of a CrmB homolog (M-T2) from myxoma disease reduced clinical indications.

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