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Comparative Study
. 2015 Jun 26;290(26):15973-84.
doi: 10.1074/jbc.M115.650119. Epub 2015 May 4.

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

Sergio M Pontejo  1 , Ali Alejo  2 , Antonio Alcami  3
Affiliations
Comparative Study

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

Sergio M Pontejo et al. J Biol Chem. .

Abstract

The blockade of tumor necrosis factor (TNF) by etanercept, a soluble version of the human TNF receptor 2 (hTNFR2), is a well established strategy to inhibit adverse TNF-mediated inflammatory responses in the clinic. A similar strategy is employed by poxviruses, encoding four viral TNF decoy receptor homologues (vTNFRs) named cytokine response modifier B (CrmB), CrmC, CrmD, and CrmE. These vTNFRs are differentially expressed by poxviral species, suggesting distinct immunomodulatory properties. Whereas the human variola virus and mouse ectromelia virus encode one vTNFR, the broad host range cowpox virus encodes all vTNFRs. We report the first comprehensive study of the functional and binding properties of these four vTNFRs, providing an explanation for their expression profile among different poxviruses. In addition, the vTNFRs activities were compared with the hTNFR2 used in the clinic. Interestingly, CrmB from variola virus, the causative agent of smallpox, is the most potent TNFR of those tested here including hTNFR2. Furthermore, we demonstrate a new immunomodulatory activity of vTNFRs, showing that CrmB and CrmD also inhibit the activity of lymphotoxin β. Similarly, we report for the first time that the hTNFR2 blocks the biological activity of lymphotoxin β. The characterization of vTNFRs optimized during virus-host evolution to modulate the host immune response provides relevant information about their potential role in pathogenesis and may be used to improve anti-inflammatory therapies based on soluble decoy TNFRs.

Keywords: TNF ligand superfamily; etanercept; immune evasion; inflammation; poxvirus; receptor; tumor necrosis factor (TNF); vTNFR; virus.

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Figures

FIGURE 1.
FIGURE 1.
Expression of recombinant vTNFRs. A, sequence alignment using Clustal Omega of the TNF binding domains of ECTV CrmD (gene E6, strain Hampstead, UniProt/TrEMBL accession no. O57300), CPXV CrmD (gene CPXV221, strain Brighton Red, UniProt/TrEMBL accession no. O57079), VARV CrmB (gene G2R, strain Bangladesh 1975, UniProt/TrEMBL accession no. P34015), CPXV CrmB (gene CPXV005, strain Brighton Red, UniProt/TrEMBL accession no. Q85308), CPXV CrmE (gene K3R, strain Elephantpox, UniProt/TrEMBL accession no. Q9DJL2), CPXV CrmC (gene CPXV191, strain Brighton Red, UniProt/TrEMBL accession no. Q9YP87), and human TNFR2 (UniProt/TrEMBL accession no. P20333). The sequences were aligned without the signal peptide and split into the different CRDs for clarity purposes. Cysteine residues are highlighted in each CRD. B, percentage of amino acid identity among the TNF binding domain of vTNFRs and hTNFR2 determined by using the web server SIAS and the substitution matrix BLOSUM62. C, schematic representation of the baculovirus expressed vTNFRs. The anti-TNFSF domain and the SECRET domain are indicated. Below, Coomassie Blue-stained gels showing 0.5–1 μg of each purified vTNFR and hTNFR2-Fc. Molecular mass is indicated in kDa.
FIGURE 2.
FIGURE 2.
Surface plasmon resonance analysis of the TNFSF members binding to vTNFRs. Four examples of the binding sensorgrams and fittings obtained for the determination of the kinetic constants of the TNFSF-vTNFR interactions: mTNF to CPXV CrmD (A), hTNF to CPXV CrmB (B), mLTα to ECTV CrmD (C), and hLTα to VARV CrmB (D). Binding and dissociation of several concentrations of TNFSF ligands at 30 μl/min were recorded and adjusted to a 1:1 Langmuir fitting (solid lines). The nanomolar concentration corresponding to each sensorgram is indicated. The arrowhead points the end of the injection.
FIGURE 3.
FIGURE 3.
TNF and LTα inhibitory activity of vTNFRs and hTNFR2. Inhibition of the cytotoxicity induced by mTNF (A), hTNF (B), mLTα (C), and hLTα (D) in L929 cells in the presence of purified recombinant vTNFRs or hTNFR2 at the indicated cytokine:protein molar ratios. Cell viability was assessed as the absorbance at 492 nm using the Cell Titer Aqueous One Solution kit (Promega). Values were normalized with the absorbance recorded from samples containing only the corresponding cytokine, and these were set to zero. Data are represented as the percentage relative to the absorbance in the absence of cytokine (media). Means ± S.D. of triplicate samples of three representative experiments are shown.
FIGURE 4.
FIGURE 4.
Inhibition of hTNF and mTNF binding to mTNFR1 by CrmE and CrmC. SPR competition experiment of 30 nm mTNF (A) and hTNF (B) binding to mTNFR1-coupled biosensor chips with increasing amounts of soluble CrmC (■) and CrmE (●). The percentage of binding refers to binding in the absence of soluble vTNFRs. RU, response units.
FIGURE 5.
FIGURE 5.
mLTβ inhibitory activity of vTNFRs. A, CrmD binding screening to mouse TNFSF by SPR. Cytokines were injected at 100 nm in HBS-EP over a ECTV CrmD coupled chip at 10 μl/min. The binding to CrmD of all commercially available mouse TNFSF members, from mTNFSF3 (mLTβ, from mLTα1β2) to mTNFSF15 (mTL1A) was recorded. As a control, mTNF was included in the screening. The arrowhead indicates the end of the injection. B and C, inhibition of the cytotoxicity induced by mLTα1β2 (B) and LTα2β1 (C) in L929 cells in the presence of purified recombinant vTNFRs and hTNFR2 at the indicated increasing cytokine:protein molar ratios. Values were normalized with the absorbance recorded from samples containing only the corresponding cytokine, and these were set to zero. Data are represented as the percentage relative to the absorbance in the absence of cytokine (media). Means ± S.D. of triplicate samples of three representative experiments are shown.
FIGURE 6.
FIGURE 6.
Summary of the inhibitory activity of vTNFRs against TNFSF members. A, vTNFR inhibitory activities diagram. The cytokines efficiently blocked by each vTNFR are indicated in the corresponding group. B, summary of the binding and inhibitory activities of all the studied vTNFRs. −, no inhibition detected; +, partial inhibition; ++, complete inhibition and low affinity (KD > 10 nm); +++, complete inhibition and high affinity (KD < 10 nm). Regarding the column of mLTβ, activities of CrmD and CrmB are referred to mLTα1β2 and mLTα2β1, respectively.
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References

    1. Aggarwal B. B. (2003) Signalling pathways of the TNF superfamily: a double-edged sword. Nat. Rev. Immunol. 3, 745–756 - PubMed
    1. Black R. A., Rauch C. T., Kozlosky C. J., Peschon J. J., Slack J. L., Wolfson M. F., Castner B. J., Stocking K. L., Reddy P., Srinivasan S., Nelson N., Boiani N., Schooley K. A., Gerhart M., Davis R., Fitzner J. N., Johnson R. S., Paxton R. J., March C. J., Cerretti D. P. (1997) A metalloproteinase disintegrin that releases tumour necrosis factor-α from cells. Nature 385, 729–733 - PubMed
    1. Browning J. L., Ngam-ek A., Lawton P., DeMarinis J., Tizard R., Chow E. P., Hession C., O'Brine-Greco B., Foley S. F., Ware C. F. (1993) Lymphotoxin β, a novel member of the TNF family that forms a heteromeric complex with lymphotoxin on the cell surface. Cell 72, 847–856 - PubMed
    1. Crowe P. D., VanArsdale T. L., Walter B. N., Ware C. F., Hession C., Ehrenfels B., Browning J. L., Din W. S., Goodwin R. G., Smith C. A. (1994) A lymphotoxin-β-specific receptor. Science 264, 707–710 - PubMed
    1. Rahman M. M., McFadden G. (2006) Modulation of tumor necrosis factor by microbial pathogens. PLoS Pathog. 2, e4. - PMC - PubMed

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