J Gen Virol. 2015 Oct;96(10):3118-23

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

Sergio M. Pontejo1,3, Ali Alejo2 and Antonio Alcami1

1Centro de Biologia Molecular Severo Ochoa (Consejo Superior de Investigaciones Cientificas-Universidad Autonoma de Madrid), 28049 Madrid, Spain.

2Centro de Investigacion en Sanidad Animal (Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria), 28130 Valdeolmos, Madrid, Spain.

3Current Address: National Institute of Allergies and Infectious Diseases (NIAID-NIH), Bethesda, MD 20892, USA.

 

Abstract

Poxviruses encode up to four different soluble viral tumor necrosis factor (TNF) receptors, named cytokine response modifier B (CrmB), CrmC, CrmD and CrmE. These proteins mimic the extracellular domain of the cellular TNF receptors to bind and inhibit the activity of TNF and, in some cases, other TNF superfamily ligands. Most of these ligands are released after the enzymatic cleavage of a membrane precursor. However, transmembrane TNF (tmTNF) is not only a precursor of soluble TNF but also exerts specific proinflammatory and immunological activities. We report that viral TNF receptors bind and inhibit tmTNF and describe some interesting differences in their activity against the soluble cytokine. Thus CrmE, which does not inhibit mouse soluble TNF, can block murine tmTNF induced cytotoxicity. We propose that this anti-tmTNF effect should be taken into consideration when assessing the role of viral TNF decoy receptors in the pathogenesis of poxvirus.

PMID: 26242179

 

Supplementary 

During thousands of years of co-evolution viruses have evolved efficient strategies to subvert the host immune system and guarantee their fitness. While small RNA viruses can rapidly incorporate mutations to escape antibody recognition, large DNA viruses such as poxviruses, can express a vast array of proteins that target key components of the immune response (1). Studying the mode of action of these poxviral immunomodulatory proteins may reveal new targets for anti-viral therapies and contribute to the understanding of the immune system mechanisms.

One of the strategies employed by poxviruses to undermine the immune response involves the expression of secreted viral proteins that specifically bind and inhibit different cytokines such as chemokines, interferons or interleukins (2). For instance, to inhibit the host tumor necrosis factor superfamily (TNFSF) ligand networks, many poxviruses encode at least one viral TNF receptor (vTNFR). vTNFRs are secreted viral proteins that mimic the ligand-binding domain of the cellular TNFRs and bind TNFSF ligands blocking their biological activities (3). Previous studies have shown that vTNFRs contribute to virulence (4, 5). Five vTNFRs have been described in poxviruses, one CD30 homologue (6), and four different TNF inhibitors termed cytokine response modifier B (CrmB), CrmC, CrmD and CrmE. Initially, CrmC and CrmE were described as specific TNF inhibitors (7, 8), whereas CrmB and CrmD inhibit both TNF and lymphotoxin a (LTa) (9, 10). We have recently identified LTb as an additional ligand for CrmB and CrmD (11).

Many ligands of the TNFSF, including TNF, are primary expressed as transmembrane proteins that after enzymatic processing release the soluble cytokine (12). However, these integral membrane cytokines are not mere precursors but can exert their own inflammatory and immune activities. While transmembrane TNF (tmTNF) can induce potent intracellular signals upon interaction with both TNF receptors, TNFR1 and TNFR2, it appears that soluble TNF (sTNF) signaling is dominated by TNFR1 (13). Therefore, tmTNF is considered the main activating ligand of TNFR2. On the other hand, the tridimensional structure of CrmE revealed that vTNFRs molecular folding contains more similarities with that of TNFR2 than of TNFR1 (14). Based on these two observations, we hypothesized that vTNFRs, classically described as inhibitors of sTNF, could block tmTNF as well.

As shown in Figure 1, the killing effect of a tmTNF-bearing mouse cell line, RAW 264.7, on the susceptible cell line, L929, was differentially blocked by increasing concentrations of recombinant vTNFRs. CrmB from variola virus, the etiologic agent of smallpox in humans, was the mos­t effective tmTNF inhibitor among the vTNFRs tested here. Interestingly, CrmE, which specifically inhibits human sTNF and fails to block mouse sTNF (11), efficiently inhibited mouse tmTNF. These results provide the first evidence of an inhibitory effect of a poxviral protein on the bioactivity of tmTNF and identify tmTNF as an additional ligand of vTNFRs.

 

 

fig1

Figure 1. vTNFRs inhibit tmTNF-induced cytotoxicity. LPS-activated mouse RAW 264.7 expressing tmTNF were co-cultured with TNF susceptible L929 cells in the presence of increasing concentrations of recombinant vTNFRs. L929 cells viability was measured 24 h later. As a negative control the SECRET domain of CrmD, which lacks of TNF binding activity, was included. vTNFRs from different poxvirus species were tested; variola virus (VARV), coxpox virus (CPXV) and ectromelia virus (ECTV).

 

Therefore, during infection, vTNFRs might inhibit both sTNF and tmTNF (Figure 2). Importantly, the interaction of tmTNF with cellular TNFRs is known to induce intracellular signals not only within TNFR expressing cells as sTNF does, but also within tmTNF-bearing cells by a process termed “reverse signaling” (15) (Figure 2, A and B). The results presented in this report demonstrate that vTNFRs inhibit the direct signaling through TNFRs induced by, besides sTNF, tmTNF and raises the interesting possibility that vTNFR might induce by reverse signaling specific cellular effects with beneficial consequences for viral fitness (Figure 2, C and D).

 

 

fig2

Figure 2. vTNFRs block the interaction of both, soluble (sTNF) and transmembrane TNF (tmTNF), with cellular TNFRs. A) In the absence of vTNFRs, tmTNF interacts with TNFRs inducing intracellular signaling within both, the TNFR- (direct signaling) and tmTNF-bearing cells (reverse signaling). B) tmTNF can be enzymatically cleaved releasing sTNF, which interacts with cellular TNFRs to induce cell signaling. C) Secreted poxviral vTNFRs bind sTNF blocking its binding to cellular TNFRs. D) vTNFR also interact with tmTNF inhibiting its direct signaling activities and potentially inducing reverse signaling within the tmTNF-bearing cells.

 

Importance of the study: Our workidentifies a novel ligand of vTNFRs, tmTNF. The fact that the cellular receptor most similar to vTNFRs is TNFR2, whose principal activating ligand is tmTNF, suggests that the membrane form of TNF could actually be the main target of vTNFRs in vivo. Although previous evidence supports a role for tmTNF in the defense against bacteria and parasites (16, 17), little is known about its relevance in the immune response upon viral infections. The study of vTNFRs in the pathogenesis of poxvirus could provide relevant information to clarify the anti-viral role of tmTNF and how poxviruses hijack this signaling network.

 

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Acknowledgements:

This work was funded by the Spanish Ministry of Economy and Competitiveness Grants SAF2009-07857 and SAF2012-38957. S.M.P. was recipient of a JAE PhD Studentship from Consejo Superior de Investigaciones Científicas and a studentship from Fundación Severo Ochoa.

 

Contact:

Antonio Alcami, PhD

Centro de Biología Molecular Severo Ochoa

Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid

Cantolanco 28049 Madrid, Spain

aalcami@cbm.csic.es

 

 

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