Engineering human interleukin-6 to obtain variants with strongly enhanced bioactivity.

Interleukin-6 (IL-6) triggers the formation of a high affinity receptor complex with the ligand binding subunit IL-6Ralpha and the signal transducing chain gp130. Since the intracytoplasmic region of the IL-6Ralpha does not contribute to signaling, soluble forms of the extracytoplasmic domain (sIL-6Ralpha), potentiate IL-6 bioactivity and induce a cytokine-responsive status in cells expressing gp130 only. This observation, together with the detection of high levels of circulating soluble human IL-6Ralpha (shIL-6Ralpha) in sera, suggests that the hIL-6-shIL-6Ralpha complex is an alternative form of the cytokine. Here we describe the generation of human IL-6 (hIL-6) variants with strongly enhanced shIL-6Ralpha binding activity and bioactivity. Homology modeling and site-directed mutagenesis of hIL-6 suggested that the binding interface for hIL-6Ralpha is constituted by the C-terminal portion of the D-helix and residues contained in the AB loop. Four libraries of hIL-6 mutants were generated by each time fully randomizing four different amino acids in the predicted AB loop. These libraries were displayed monovalently on filamentous phage surface and sorted separately for binding to immobilized shIL-6Ralpha. Mutants were selected which, when expressed as soluble proteins, showed a 10- to 40-fold improvement in shIL-6Ralpha binding; a further increase (up to 70-fold) was achieved by combining variants isolated from different libraries. Interestingly, high affinity hIL-6 variants show strongly enhanced bioactivity on cells expressing gp13O in the presence of shIL-6Ralpha at concentrations similar to those normally found in human sera.     

IL-4 receptor alpha is an important modulator of IL-4 and IL-13 receptor binding: implications for the development of therapeutic targets

IL-4 is a key cytokine associated with allergy and asthma. Induction of cell signaling by IL-4 involves interaction with its cognate receptors, a complex of IL-4Ralpha with either the common gamma-chain or the IL-13R chain alpha1 (IL-13Ralpha1). We found that IL-4 bound to the extracellular domain of IL-4Ralpha (soluble human (sh)IL-4Ralpha) with high affinity and specificity. In contrast with the sequential mechanism of binding and stabilization afforded by IL-4Ralpha to the binding of IL-13 to IL-13Ralpha1, neither common gamma-chain nor IL-13Ralpha1 contributed significantly to the stabilization of the IL-4:IL-4Ralpha complex. Based on the different mechanisms of binding and stabilization of the IL-4R and IL-13R complexes, we compared the effects of shIL-4Ralpha and an IL-4 double mutein (R121D/Y124D, IL-4R antagonist) on IL-4- and IL-13-mediated responses. Whereas IL-4R antagonist blocked responses to both cytokines, shIL-4Ralpha only blocked IL-4. However, shIL-4Ralpha stabilized and augmented IL-13-mediated STAT6 activation and eotaxin production by primary human bronchial fibroblasts at suboptimal doses of IL-13. These data demonstrate that IL-4Ralpha plays a key role in the binding affinity of both IL-13R and IL-4R complexes. Under certain conditions, shIL-4Ralpha has the potential to stabilize binding IL-13 to its receptor to augment IL-13-mediated responses. Thus, complete understanding of the binding interactions between IL-4 and IL-13 and their cognate receptors may facilitate development of novel treatments for asthma that selectively target these cytokines without unpredicted or detrimental side effects.     

Characterization of the interaction between interleukin-13 and interleukin-13 receptors

Interleukin-13 (IL-13) possesses two types of receptor: the heterodimer, composed of the IL-13Ralpha1 chain (IL-13Ralpha1) and the IL-4Ralpha chain (IL-4Ralpha), transducing the IL-13 signals; and the IL-13Ralpha2 chain (IL-13Ralpha2), acting as a nonsignaling "decoy" receptor. Extracellular portions of both IL-13Ralpha1 and IL-13Ralpha2 are composed of three fibronectin type III domains, D1, D2, and D3, of which the last two comprise the cytokine receptor homology modules (CRHs), a common structure of the class I cytokine receptor superfamily. Thus far, there has been no information about the critical amino acids of the CRHs or the role of the D1 domains of IL-13Ralpha1 and IL-13Ralpha2 in binding to IL-13. In this study, we first built the homology modeling of the IL-13.hIL-13 receptor complexes and then predicted the amino acids involved in binding to IL-13. By incorporating mutations into these amino acids, we identified Tyr-207, Asp-271, Tyr-315, and Asp-318 in the CRH of human IL-13Ralpha2, and Leu-319 and Tyr-321 in the CRH of human IL-13Ralpha1, as critical residues for binding to IL-13. Tyr-315 in IL-13Ralpha2 and Leu-319 in IL-13Ralpha1 are positionally conserved hydrophobic amino acid residues. Furthermore, by using D1 domain-deleted mutants, we found that the D1 domain is needed for the expression of IL-13Ralpha2, but not IL-13Ralpha1, and that the D1 domain of IL-13Ralpha1 is important for binding to IL-13, but not to IL-4. These results provide the basis for a precise understanding of the interaction between IL-13 and its receptors.     

Molecular cloning of the guinea-pig IL-5 receptor alpha and beta subunits and reconstitution of a high affinity receptor

The functional IL-5 receptor is a heteromeric complex consisting of an alpha and beta subunit. The cloning, sequencing and expression of guinea-pig IL-5Ralpha and beta subunits is described. The guinea-pig IL-5Ralpha subunit cDNA encodes a protein of M(r)47 kDa, which is 72 and 66% homologous to the human and murine orthologs, respectively. Three guinea-pig IL-5Rbeta subunit cDNA clones were isolated, which differ in the N-terminus and are 56-64% homologous to the human and murine IL-5Rbeta subunits. Expressing human IL-5Ralphabeta and guinea-pig IL-5Ralphabeta(1)in the baculovirus-insect cell system resulted in recombinant receptors which bound hIL-5 with high affinity (K(d)=0.19 and 0.11 nM, respectively). Expressing just gpIL-5Ralpha was not sufficient to demonstrate binding. This contrasts with the human receptor, where hIL-5Ralpha alone can bind hIL-5 with high affinity. gpIL-5Ralphabeta(1)bound both hIL-5 and mIL-5 with comparable affinity (K(i)=0.10 and 0.06 nM), similar to that seen with hIL-5Ralphabeta. Thus, both the heteromeric hIL-5R and gpIL-5Ralphabeta(1)can bind multiple IL-5 orthologs with high affinity whereas the murine IL-5R is selective for the murine ligand.     

Expression of human IL-13 receptor alpha2 extracellular domain in Pichia pastoris

Interleukin-13 receptor alpha2 (IL-13Ralpha2) binds IL-13 with high affinity and plays an important role in IL-13 signaling as a decoy receptor. We expressed the extracellular domain of human IL-13Ralpha2 (1-313) in methylotrophic yeast Pichia pastoris. SDS-PAGE analysis by PAS staining and Western blot analysis detected the product of the extracellular domain of human IL-13Ralpha2 as glycoprotein from P. pastoris. The yield of purified extracellular domain of human IL-13Ralpha2 was 2mg from 1L of culture. From CD analysis, the 2D structure of the purified IL-13Ralpha2 showed the typical beta-sheet. ELISA of the purified IL-13Ralpha2 detected the binding activity for human IL-13. Thus, it was found that the active extracellular domain of human IL-13Ralpha2 was expressed from P. pastoris.     

A novel mechanism by which interferon-gamma can regulate interleukin (IL)-13 responses. Evidence for intracellular stores of IL-13 receptor alpha -2 and their rapid mobilization by interferon-gamma.

Interleukin (IL)-13 mediates its activities via a complex receptor system. Interleukin-13 receptor alpha-1 chain (IL-13Ralpha1) binds IL-13 with low affinity, but does not signal. However, when IL-13Ralpha1 combines with IL-4 receptor alpha (IL-4Ralpha), a signaling high affinity receptor complex for IL-13 is generated. In contrast, IL-13Ralpha2 alone binds IL-13 with high affinity, but does not signal and has been postulated to be a decoy receptor. Herein, we investigated the cellular localization of IL-13Ralpha2 and the regulation of its expression by confocal microscopy and flow cytometry in primary and cultured cells. Our results demonstrate that IL-13Ralpha2 is largely an intracellular molecule, which is rapidly mobilized from intracellular stores following treatment with interferon (IFN)-gamma. Up-regulation of IL-13Ralpha2 surface expression in response to IFN-gamma was rapid, did not require protein synthesis, and resulted in diminished IL-13 signaling. These results provide the first evidence that the IL-13Ralpha2 is predominantly an intracellular molecule and demonstrate a novel mechanism by which IFN-gamma can regulate IL-13 responses.     

On-column refolding and characterization of soluble human interleukin-15 receptor alpha-chain produced in Escherichia coli

Interleukin-15 receptor alpha-chain (IL-15Ralpha) is a member of the new cytokine receptor family, which possesses the sushi domain. To investigate the biochemical and biophysical characteristics of soluble human IL-15Ralpha (shIL-15Ralpha), shIL-15Ralpha was recombinantly expressed in Escherichia coli. The shIL-15Ralpha containing a six histidine-tag was expressed as inclusion bodies, which were solubilized with urea, immobilized on a Ni-nitrilotriacetic acid column, and refolded by a decreasing gradient of urea concentration. The refolded shIL-15Ralpha exhibited a highly flexible structure, neutralized human interleukin-15-induced cell proliferation effectively, and bound to its ligand with the same affinity as human IL-15Ralpha on the cell surface, as demonstrated by circular dichroism, a cell proliferation assay, and surface plasmon resonance, respectively. Thus, we succeeded in refolding shIL-15Ralpha to an active form on an affinity column.     

Mechanism of action of interleukin-13 antagonist (IL-13E13K) in cells expressing various types of IL-4R

As interleukin (IL)-13 and IL-4 play a major role in various diseases including asthma, allergy, and malignancies, it is desirable to generate a molecule that blocks the effects of both cytokines. We previously generated a human IL-13 mutant (IL-13E13K), which is a powerful antagonist of IL-13, blocking the biological activities of IL-13. We now show that IL-13E13K also competitively inhibits signaling and biological activities of IL-4 through type II and partially through type III IL-4 receptor (R) system. IL-13E13K completely blocked the IL-4-induced phosphorylation of STAT6 and IL-4-dependent protein synthesis in cells expressing type II and partially type III IL-4R but not type I IL- 4R. Consistent with the inhibition of biological activities, IL-13E13K inhibited IL-4 binding to type II IL-4R-expressing cells but not to type I IL-4R-expressing cells. The inhibition efficiency of IL-4 binding by IL-13E13K was relatively lower compared to wtIL-13 even though IL-13E13K bound to IL-13Ralpha1 positive cells with a similar affinity to wtIL-13. These results indicate that Glu13 in IL-13 associates with IL-4Ralpha, and mutation to lysine decreases its binding ability to IL-4Ralpha chain. IL-13E13K binds to IL- 13Ralpha1, which is shared by both IL-13R and IL-4R systems. Consequently, IL-13E13K inhibits IL-4 binding to these cells and prevents heterodimer formation between IL-13Ralpha1 and IL-4Ralpha chains. This interference by IL-13E13K blocks the biological activities of not only IL-13 but also partially of IL-4. Thus, IL-13E13K may be a useful agent for the treatment of diseases such as asthma, allergic rhinitis, and cancer, which are dependent on signaling through both IL-4 and IL-13 receptors.     

Purification of receptor complexes of interleukin-10 stoichiometry and the importance of deglycosylation in their crystallization.

Interleukin-10 (IL-10) is a pleiotropic immunosuppressive cytokine that has a wide range of effects in controlling inflammatory responses. Viral IL-10 (vIL-10) is a homologue of human IL-10 (hIL-10) produced by Epstein-Barr virus (EBV). Both hIL-10 and vIL-10 bind to the soluble extracellular fragment of the cytokine receptor IL-10R1 (shIL-10R1). The stoichiometry of the vIL-10 : shIL-10R1 complex has been found to be the same as hIL-10 : shIL-10R1, with two vIL-10 dimers binding to four shIL-10R1 monomers. Complexes of both hIL-10 and vIL-10 with glycosylated shIL-10R1 could not be crystallized. Controlled deglycosylation using peptide : N-glycosidase F and endo-beta-N-acetylglucosaminidase F3 resulted in the formation of crystals of both hIL-10 : shIL-10R1 and vIL-10 : shIL-10R1 complexes, indicating that the difficulty in the crystal formation was largely due to the presence of complex carbohydrate side chains. The availability of the structure of the ligand-receptor complexes should facilitate our understanding of the basis of the interaction between IL-10 and the IL-10 receptor.     

Expression cloning of the human IL-3 receptor cDNA reveals a shared beta subunit for the human IL-3 and GM-CSF receptors.

A cDNA for a human interleukin-3 (hIL-3) binding protein has been isolated by a novel expression cloning strategy: a cDNA library was coexpressed with the cDNA for the beta subunit of human granulocyte/macrophage colony-stimulating factor (GM-CSF) receptor (hGMR beta) in COS7 cells and screened by binding of 125I-labeled IL-3. The cloned cDNA (DUK-1) encodes a mature protein of 70 kd, which belongs to the cytokine receptor family and which alone binds hIL-3 with extremely low affinity (Kd = 120 +/- 60 nM). A high affinity IL-3-binding site (Kd = 140 +/- 30 pM) was reconstituted by coexpressing the DUK-1 protein and hGMR beta, indicating that hIL-3R and hGMR share the beta subunit. Therefore, we designated DUK-1 as the alpha subunit of the hIL-3R. As in human hematopoietic cells, hIL-3 and hGM-CSF complete for binding in fibroblasts expressing the cDNAs for hIL-3R alpha, GMR alpha, and the common beta subunit, indicating that different alpha subunits compete for a common beta subunit.     

Immunoreceptor tyrosine-based inhibitory motif of the IL-4 receptor associates with SH2-containing phosphatases and regulates IL-4-induced proliferation.

Immunoreceptor tyrosine-based inhibitory motifs (ITIM) have been implicated in the negative modulation of immunoreceptor signaling pathways. The IL-4R alpha-chain (IL-4Ralpha) contains a putative ITIM in the carboxyl terminal. To determine the role of ITIM in the IL-4 signaling pathway, we ablated the ITIM of IL-4Ralpha by deletion and site-directed mutagenesis and stably expressed the wild-type (WT) and mutant hIL-4Ralpha in 32D/insulin receptor substrate-2 (IRS-2) cells. Strikingly, 32D/IRS-2 cells expressing mutant human (h)IL-4Ralpha were hyperproliferative in response to IL-4 compared with cells expressing WT hIL-4Ralpha. Enhanced tyrosine phosphorylation of Stat6, but not IRS-2, induced by hIL-4 was observed in cells expressing mutant Y713F. Using peptides corresponding to the ITIM of hIL-4Ralpha, we demonstrate that tyrosine-phosphorylated peptides, but not their nonphosphorylated counterparts, coprecipitate SH2-containing tyrosine phosphatase-1, SH2-containing tyrosine phosphatase-2, and SH2-containing inositol 5'-phosphatase. The in vivo association of SH2-containing inositol 5'-phosphatase with IL-4Ralpha was verified by coimmunoprecipitation with anti-IL-4Ralpha Abs. These results demonstrate a functional role for ITIM in the regulation of IL-4-induced proliferation.     

Analysis of the role of the interleukin-2 receptor gamma chain in ligand binding

Interleukin-2 is the primary T cell growth factor secreted by activated T cells. IL-2 is an alpha-helical cytokine that binds to a multisubunit receptor expressed on the surface of a variety of cell types. IL-2Ralpha, IL-2Rbeta, and IL-2Rgammac receptor subunits expressed on the surface of cells may aggregate to form distinct binding sites of differing affinities. IL-2Rgammac was the last receptor subunit to be identified. It has since been shown to be shared by at least five other cytokine receptors. In this study, we have probed the role of IL-2Rgammac in the assembly of IL-2R complexes and in ligand binding. We demonstrate that in the absence of ligand IL-2Rgammac does not possess detectable affinity for IL-2Ralpha, IL-2Rbeta, or the pseudo-high-affinity binding site composed of preformed IL-2Ralpha/beta. We also demonstrate that IL-2Rgammac possesses an IL-2-dependent affinity for IL-2Rbeta and IL-2Ralpha/beta. We performed a detailed biosensor analysis to examine the interaction of soluble IL-2Rgammac with IL-2-bound IL-2Rbeta and IL-2-bound IL-2Ralpha/beta. The kinetic and equilibrium constants for sIL-2Rgammac binding to these two different liganded complexes were similar, indicating that IL-2Ralpha does not play a role in recruitment of IL-2Rgammac. We also determined that the binding of IL-2 to the isolated IL-2Rgammac was very weak (approximate K(D) = 0.7 mM). The experimental methodologies and principles derived from these studies can be extended to at least five other cytokines that share IL-2Rgammac as a receptor subunit.     

Level of expression of IL-13R alpha 2 impacts receptor distribution and IL-13 signaling

IL-13, a critical cytokine for allergic inflammation, exerts its effects through a complex receptor system including IL-4Ralpha, IL-13Ralpha1, and IL-13Ralpha2. IL-4Ralpha and IL-13Ralpha1 form a heterodimeric signaling receptor for IL-13. In contrast, IL-13Ralpha2 binds IL-13 with high affinity but does not signal. IL-13Ralpha2 exists on the cell surface, intracellularly, and in soluble form, but no information is available regarding the relative distributions of IL-13Ralpha2 among these compartments, whether the compartments communicate, and how the relative expression levels impact IL-13 responses. Herein, we investigated the distribution of IL-13Ralpha2 in transfected and primary cells, and we evaluated how the total level of IL-13Ralpha2 expression impacted its distribution. Our results demonstrate that the distribution of IL-13Ralpha2 is independent of the overall level of expression. The majority of the IL-13Ralpha2 protein existed in intracellular pools. Surface IL-13Ralpha2 was continually released into the medium in a soluble form, yet surface expression remained constant supporting receptor trafficking to the cell surface. IL-13Ralpha2 inhibited IL-13 signaling proportionally to its level of expression, and this inhibition could be overcome with high concentrations of IL-13.     

Receptors for interleukin-13 and interleukin-4 are complex and share a novel component that functions in signal transduction.

Interleukin-4 (IL-4) and interleukin-13 (IL-13) are two cytokines that are secreted by activated T cells and have similar effects on monocytes and B cells. We describe a mutant form of human interleukin-4 (hIL-4) that competitively antagonizes both hIL-4 and human interleukin-13 (hIL-13). The amino acid sequences of IL-4 and IL-13 are approximately 30% homologous and circular dichroism (CD) spectroscopy shows that both proteins have a highly alpha-helical structure. IL-13 competitively inhibited binding of hIL-4 to functional human IL-4 receptors (called hIL-4R) expressed on a cell line which responds to both hIL-4 and IL-13. Binding of hIL-4 to an hIL-4 responsive cell line that does not respond to IL-13, and binding of hIL-4 to cloned IL-4R ligand binding protein expressed on heterologous cells, were not inhibited by IL-13. hIL-4 bound with approximately 100-fold lower affinity to the IL-4R ligand binding protein than to functional IL-4R. The mutant hIL-4 antagonist protein bound to both IL-4R types with the lower affinity. The above results demonstrate that IL-4 and IL-13 share a receptor component that is important for signal transduction. In addition, our data establish that IL-4R is a complex of at least two components one of which is a novel affinity converting subunit that is critical for cellular signal transduction.     

Stat6 is necessary and sufficient for IL-4's role in Th2 differentiation and cell expansion

IL-4 plays a critical role in the differentiation of TCR-stimulated naive CD4 T cells to the Th2 phenotype. In response to IL-4, the IL-4R activates a set of phosphotyrosine binding domain-containing proteins, including insulin receptor substrate 1/2, Shc, and IL-4R interacting protein, as well as Stat6. Stat6 has been shown to be required for Th2 differentiation. To determine the roles of the phosphotyrosine binding adaptors in Th2 differentiation, we prepared a retrovirus containing a mutant of the human (h)IL-4R alpha-chain, Y497F, which is unable to recruit these adaptors. The mutant hIL-4Ralpha, as well as the wild-type (WT) hIL-4Ralpha, was introduced into naive CD4 T cells. Upon hIL-4 stimulation, Y497F worked as well as the WT hIL-4Ralpha in driving Th2 differentiation, as measured by Gata3 up-regulation and IL-4 production. Furthermore, IL-4-driven cell expansion was also normal in the cells infected with Y497F, although cells infected with Y497F were not capable of phosphorylating insulin receptor substrate 2. These results suggest that the signal pathway mediated by Y497 is dispensable for both IL-4-driven Th2 differentiation and cell expansion. Both WT and Y497F hIL-4Ralpha lose the ability to drive Th2 differentiation and cell expansion in Stat6-knockout CD4 T cells. A constitutively activated form of Stat6 introduced into CD4 T cells resulted in both Th2 differentiation and enhanced cell expansion. Thus, activated Stat6 is necessary and sufficient to mediate both IL-4-driven Th2 differentiation and cell expansion in CD4 T cells.     

IL-6, leukemia inhibitory factor,and oncostatin M stimulate bone resorption and regulate the expression of receptor activator of NF-kappa B ligand,osteoprotegerin, and receptor activator of NF-kappa B in mouse calvariae

IL-6, leukemia inhibitory factor (LIF), and oncostatin M (OSM) are IL-6-type cytokines that stimulate osteoclast formation and function. In the present study, the resorptive effects of these agents and their regulation of receptor activator of NF-kappaB ligand (RANKL), RANK, and osteoprotegerin (OPG) were studied in neonatal mouse calvaria. When tested separately, neither human (h) IL-6 nor the human soluble IL-6R (shIL-6R) stimulated bone resorption, but when hIL-6 and the shIL-6R were combined, significant stimulation of both mineral and matrix release from bone explants was noted. Semiquantitative RT-PCR showed that hIL-6 plus shIL-6R enhanced the expression of RANKL and OPG in calvarial bones, but decreased RANK expression. Human LIF, hOSM, and mouse OSM (mOSM) also stimulated 45Ca release and enhanced the mRNA expression of RANKL and OPG in mouse calvaria, but had no effect on the expression of RANK. In agreement with the RT-PCR analyses, ELISA measurements showed that both hIL-6 plus shIL-6R and mOSM increased RANKL and OPG proteins. 1,25-Dihydroxyvitamin D3 (D3) also increased the RANKL protein level, but decreased the protein level of OPG. OPG inhibited 45Ca release stimulated by RANKL, hIL-6 plus shIL-6R, hLIF, hOSM, mOSM, and D3. An Ab neutralizing mouse gp130 inhibited 45Ca release induced by hIL-6 plus shIL-6R. These experiments demonstrated stimulation of calvarial bone resorption and regulation of mRNA and protein expression of RANKL and OPG by D3 and IL-6 family cytokines as well as regulation of RANK expression in preosteoclasts/osteoclasts of mouse calvaria by D3 and hIL-6 plus shIL-6R.     

Identification of regions within the third FnIII-like domain of the IL-5Ralpha involved in IL-5 interaction

Previously, two binding sites for interleukin 5 (IL-5) were identified on the IL-5 receptor alpha chain (IL-5Ralpha). They are located within the CD loop of the first fibronectin type III (FnIII)-like domain and the EF loop of the second FnIII-like domain. The first binding site was identified by exploiting the different abilities of human IL-5Ralpha (hIL-5Ralpha) and mouse IL-5Ralpha (mIL-5Ralpha) to bind hIL-5. Here we show that ovine IL-5 (oIL-5) has the ability to activate the hIL-5Ralpha but not the mIL-5Ralpha. By using chimeras of the mIL-5Ralpha and hIL-5Ralpha we demonstrate that residues within the first and third FnIII-like domains of mIL-5Ralpha are responsible for this lack of activity. Furthermore, mutation of residues on hIL-5Ralpha to mIL-5Ralpha within the predicted DE and FG loop regions of the third FnIII domain reduces oIL-5 activity. These results show that regions of the third FnIII domain of IL-5Ralpha are involved in binding, in addition to the regions in domains one and two of the IL-5Ralpha that were identified in an earlier study.     

Reversal of some viral IL-6 electrostatic properties compared to IL-6 contributes to a loss of alpha receptor component recruitment

Human interleukin-6 (hIL-6) is a pleiotropic mediator of activation and proliferation across a large number of different cell types. Human herpesvirus-8 (HHV-8) has been associated with classical and AIDS-related Kaposi's sarcoma (KS). HHV-8 encodes viral IL-6 (vIL-6), a functional homolog of human interleukin-6, that promotes the growth of KS and of some lymphoma cells. Signaling induced by human IL-6 requires recruitment of the glycoprotein gp130, which acts as the signal transducing chain, and of IL-6Ralpha, which is necessary for cognate recognition and high affinity receptor complex formation. In contrast, the formation of a functional complex between vIL-6 and gp130 does not require the presence of IL-6Ralpha. The physico-chemical properties of vIL-6 have been analyzed and compared to those of hIL-6 and of the receptor chains, gp130 and IL-6Ralpha. Interaction sites on vIL-6 involve more hydrophobic residues than those of hIL-6. The electrostatic fields induced by vIL-6 and IL-6Ralpha are repulsive and prevent interaction between vIL-6 and IL-6Ralpha, whereas the electrostatic field induced by hIL-6 steers the complex formation with IL-6Ralpha. Subsequently, electrostatic binding free energy in the vIL-6/IL-6Ralpha complex is destabilizing, whereas it is stabilizing in the complex comprising hIL-6. These properties result from charge reversals between viral and human IL-6, an unusual phenomenon of amino acid substitutions within a homologous protein family. This suggests a selection pressure for vIL-6 to by-pass the IL-6Ralpha control of host defense against virus infection. This selection pressure has yielded the reversal of electrostatic properties of vIL-6 when compared to hIL-6.