Insights into ligand binding by a viral tumor necrosis factor (TNF) decoy receptor yield a selective soluble human type 2 TNF receptor.

Etanercept is a soluble form of the tumor necrosis factor receptor 2 (TNFR2) that inhibits pathological tumor necrosis factor (TNF) responses in rheumatoid arthritis and other inflammatory diseases. However, besides TNF, etanercept also blocks lymphotoxin-α (LTα), which has no clear therapeutic value and might aggravate some of the adverse effects associated with etanercept. Poxviruses encode soluble TNFR2 homologs, termed viral TNF decoy receptors (vTNFRs), that display unique specificity properties. For instance, cytokine response modifier D (CrmD) inhibits mouse and human TNF and mouse LTα, but it is inactive against human LTα. Here, we analyzed the molecular basis of these immunomodulatory activities in the ectromelia virus-encoded CrmD. We found that the overall molecular mechanism to bind TNF and LTα from mouse and human origin is fairly conserved in CrmD and dominated by a groove under its 50s loop. However, other ligand-specific binding determinants optimize CrmD for the inhibition of mouse ligands, especially mouse TNF. Moreover, we show that the inability of CrmD to inhibit human LTα is caused by a Glu-Phe-Glu motif in its 90s loop. Importantly, transfer of this motif to etanercept diminished its anti-LTα activity in >60-fold while weakening its TNF-inhibitory capacity in 3-fold. This new etanercept variant could potentially be used in the clinic as a safer alternative to conventional etanercept. This work is the most detailed study of the vTNFR-ligand interactions to date and illustrates that a better knowledge of vTNFRs can provide valuable information to improve current anti-TNF therapies.

Mechanism of action of the viral chemokine-binding protein E163 from ectromelia virus.

Chemokines interact with glycosaminoglycans (GAGs) at the cellular surface and to specific cell-surface receptors to activate signaling pathways. The GAG interaction allows the formation of a chemotactic gradient of chemokine required for cell haptotaxis and chemokine oligomerization. Poxviruses encode secreted chemokine-binding proteins with no sequence similarity to their cellular counterparts to modulate the host immune system. The E163 protein from ectromelia virus, the causative agent of mousepox, binds chemokines through their GAG-binding domain. In addition, E163 interacts with GAGs to be anchored at the cell surface, but its ability to interfere with chemokine-GAG interactions has not been demonstrated. We report the identification of the GAG-binding regions in E163 and the generation of mutant forms deficient of GAG binding. Chemokine binding assays show that some of the E163 GAG-binding sites are also involved in the interaction with chemokines. By using recombinant GAG-binding mutant forms we demonstrate that E163 prevents the interaction of chemokines with cell-surface GAGs, providing mechanisms for the immunomodulatory activity of the viral chemokine-binding protein E163.

Structural basis for antagonism of human interleukin 18 by poxvirus interleukin 18-binding protein.

Human interleukin-18 (hIL-18) is a cytokine that plays an important role in inflammation and host defense against microbes. Its activity is regulated in vivo by a naturally occurring antagonist, the human IL-18-binding protein (IL-18BP). Functional homologs of human IL-18BP are encoded by all orthopoxviruses, including variola virus, the causative agent of smallpox. They contribute to virulence by suppressing IL-18-mediated immune responses. Here, we describe the 2.0-A resolution crystal structure of an orthopoxvirus IL-18BP, ectromelia virus IL-18BP (ectvIL-18BP), in complex with hIL-18. The hIL-18 structure in the complex shows significant conformational change at the binding interface compared with the structure of ligand-free hIL-18, indicating that the binding is mediated by an induced-fit mechanism. EctvIL-18BP adopts a canonical Ig fold and interacts via one edge of its beta-sandwich with 3 cavities on the hIL-18 surface through extensive hydrophobic and hydrogen bonding interactions. Most of the ectvIL-18BP residues that participate in these interactions are conserved in both human and viral homologs, explaining their functional equivalence despite limited sequence homology. EctvIL-18BP blocks a putative receptor-binding site on IL-18, thus preventing IL-18 from engaging its receptor. Our structure provides insights into how IL-18BPs modulate hIL-18 activity. The revealed binding interface provides the basis for rational design of inhibitors against orthopoxvirus IL-18BP (for treating orthopoxvirus infection) or hIL-18 (for treating certain inflammatory and autoimmune diseases).

Crystal structures of a poxviral glutaredoxin in the oxidized and reduced states show redox-correlated structural changes.

Glutaredoxins act as reducing agents for the large subunit of ribonucleotide reductase (R1) in many prokaryotes and eukaryotes, including humans. The same relationship has been proposed for the glutaredoxin and R1 proteins expressed by all orthopoxviruses, including vaccinia, variola, and ectromelia virus. Interestingly, the orthopoxviral proteins share 45% and 78% sequence identity with human glutaredoxin-1 (Grx-1) and R1, respectively. To study structure-function relationships of the vertebrate Grx-1 family, and reveal potential viral adaptations, we have determined crystal structures of the ectromelia virus glutaredoxin, EVM053, in the oxidized and reduced states. The structures show a large redox-induced conformational rearrangement of Tyr21 and Thr22 near the active site. We predict that the movement of Tyr21 is a viral-specific adaptation that increases the redox potential by stabilizing the reduced state. The conformational switch of Thr22 appears to be shared by vertebrate Grx-1 and may affect the strictly conserved Lys20. A crystal packing-induced structural change in residues 68-70 affects the GSH-binding loop, and our structures reveal a potential interaction network that connects the GSH-binding loop and the active site. EVM053 also exhibits a novel cis-proline (Pro53) in a loop that has been shown to contribute to R1-binding in Escherichia coli Grx-1. The cis-peptide bond of Pro53 may be required to promote electrostatic interactions between Lys52 and the C-terminal carboxylate of R1. Finally, dimethylarsenite was covalently attached to Cys23 in one reduced EVM053 structure and our preliminary data show that EVM053 has dimethylarsenate reductase activity.

Identification of residues in the ectromelia virus gamma interferon-binding protein involved in expanded species specificity.

Gamma interferon (IFN-gamma) production is important in the host response to, and recovery from, infection with Ectromelia virus (ECTV) and Vaccinia virus (VACV). The orthopoxviruses have evolved several mechanisms to subvert the IFN-gamma response. IFN-gamma-binding protein (IFN-gammaBP) is a virally encoded homologue of the host IFN-gamma receptor that blocks the effects of IFN-gamma in the infected host. Unlike the cellular receptors, whose ligand specificity is restricted to their own species, the orthopoxvirus IFN-gammaBPs bind IFN-gamma from several species. The reason for this relaxed specificity has yet to be explained. ECTV, a mouse pathogen, encodes an IFN-gammaBP that has been shown to inhibit the activity of both human and murine IFN-gamma (hIFN-gamma and mIFN-gamma, respectively). In contrast, the IFN-gammaBP from VACV is unable to inhibit mIFN-gamma, but retains activity against hIFN-gamma. To determine which region(s) in the ECTV sequence is responsible for its ability to bind to mIFN-gamma with high affinity, a series of chimeric IFN-gammaBPs, as well as individual point mutants in the ECTV sequence corresponding to the amino acid changes from the VACV sequence, were constructed. The affinities of the chimeric and point mutant IFN-gammaBPs for mIFN-gamma were tested by using surface plasmon resonance and bioassay. By using this strategy, several key residues in the ligand-binding domains of the ECTV sequence have been identified that are responsible for high-affinity binding to mIFN-gamma. Substitution of the ECTV residue at these positions in VACV resulted in a dramatic increase in the affinity of the VACV IFN-gammaBP for mIFN-gamma.

[Fusion proteins encoded by orf 129L of ectromelia and orf A30L of smallpox viruses cross-react with neutralizing monoclonal antibodies].

Open reading frame (orf) 129L of ectromelia (EV) and orf A30L of smallpox viruses (SPV) encoding fusion proteins were cloned and expressed in E. coli cells. The recombinant polypeptides (prA30L H pr129L) were purified from cell lysates by Ni-NTA chromatography. Recombinant polypeptides were able to form trimers in buffered saline and they destroyed under treatment with SDS and 2-mercaptoethanol. Reactivity of prA30L, pr129L and orthopoxvirus proteins was analyzed by ELISA and Western blotting with panel of 22 monoclonal antibodies (MAbs) against orthopoxviruses (19 against EV, 2 MAbs against vaccinia virus and 1 Mabs against cowpox virus). This data allowed us to conclude that there are 12 EV-specific epitopes of pr129L and EV fusion proteins, ten orthopox-specific epitopes of EV, VV, CPV fusion proteins, from them 9 orthopox-specific epitopes of prA30L and SPV fusion proteins. Five Mabs, which cross-reacted with orthopox-specific epitopes, were able to neutralize the VV on Vero cells and from them two MAbs has neutralizing activity against smallpox virus. Our findings demonstrate that 129L fusion protein have EV-specific epitopes, that EV 129L and SPV A30L fusion proteins have a several orthopox-specific epitopes to induce a neutralizing antibodies against human pathogenic orthopoxviruses.

Purification, crystallization and preliminary diffraction studies of an ectromelia virus glutaredoxin.

Ectromelia, vaccinia, smallpox and other closely related viruses of the orthopoxvirus genus encode a glutaredoxin gene that is not present in poxviruses outside of this genus. The vaccinia glutaredoxin O2L has been implicated as the reducing agent for ribonucleotide reductase and may thus play an important role in viral deoxyribonucleotide synthesis. As part of an effort to understand nucleotide metabolism by poxviruses, EVM053, the O2L ortholog of the ectromelia virus, has been crystallized. EVM053 crystallizes in space group C222(1), with unit-cell parameters a = 61.98, b = 67.57, c = 108.55 A. Diffraction data have been processed to 1.8 A resolution and a self-rotation function indicates that there are two molecules per asymmetric unit.

Characterization of the ectromelia virus serpin, SPI-2.

Poxviruses encode multiple proteins that enable them to evade host responses. Among these are serine protease inhibitors (serpins). One of the earliest serpins described, cowpox virus crmA, acts to inhibit inflammation and apoptosis. crmA homologous serpins, known as SPI-2, are conserved in rabbitpox, vaccinia and variola viruses. Here, we describe the characterization of ectromelia virus (EV) SPI-2. EV SPI-2 encodes a protein of approximately 38 kDa showing >94% identity with other poxviral homologues. Conservative changes in amino acid sequence were found within the reactive site loop and the serpin backbone. Like crmA, transient expression of SPI-2 protected cells from tumour necrosis factor-mediated apoptosis and inhibited the activity of caspases-1 and -8 but not caspases-3, -6 or granzyme B. Overall, this study demonstrates that EV SPI-2 is functionally similar to crmA, based on in vitro assays.

Analysis of host response modifier ORFs of ectromelia virus, the causative agent of mousepox.

From the right-hand end of the ectromelia virus (strain Moscow) genome, 32318 bps have been sequenced, and characterized to include a total of 18 open reading frames (ORFs) and six regions which apparently no longer code for functional proteins. At least six of the ORFs appear to be involved in blocking the inflammatory/immune host response to infection, and therefore probably contribute significantly to the virulence of this virus in its natural host, the mouse. One of these genes encoded an isolog of the poxvirus chemokine binding protein, and was shown to be the most abundant protein secreted from ectromelia virus infected cells. Two regions were found to have significant similarity to poxvirus genes encoding tumor necrosis factor (TNF) binding proteins. Both are distinct from cytokine response modifier (crm)B and crmC but only one is predicted to encode a functional TNF binding protein. A novel similarity between the C-terminal domain of poxvirus TNF binding proteins and several other poxvirus proteins is also presented. The results are discussed in the context of ectromelia virus pathogenesis of mice.

A poxvirus protein with a RING zinc finger motif is of crucial importance for virulence.

The DNA sequence of a 2060-bp fragment from the left-hand end of the ectromelia (mousepox) virus genome was determined. Two genes were identified coding for 28 kDa (p28) and 16 kDa (p16) proteins, both of which are disrupted in vaccinia virus but conserved in variola (smallpox) virus. p16 contains an N-terminal hydrophobic region and may be a membrane or secreted protein. p28 contains a C3HC4 (RING) zinc finger motif that has been found in a large family of proteins involved mostly in transcription regulation. p28 was expressed in bacteria and shown to bind Zn in vitro. Disruption of the p28 gene had no appreciable effect on the multiplication of the virus in cell culture but abolished its lethality for susceptible mice. The p28- mutant virus replicated to significantly lower titers than the wild-type virus in different organs of infected mice. It is proposed that the p28 gene is an important determinant of orthopoxvirus pathogenicity, and its product may positively or negatively regulate expression of host or viral gene(s) involved in virus-host interaction.