Detection of Direct Binding of Human Herpesvirus 8-Encoded Interleukin-6 (vIL-6) to both gp130 and IL-6 Receptor (IL-6R) and Identification of Amino Acid Residues of vIL-6 Important for IL-6R-Dependent and -Independent Signaling

Human herpesvirus 8 (HHV-8) has been associated with all forms of Koposi's sarcoma (KS) and primary effusion lymphoma (PEL) and is found in a high proportion of affected lymph nodes of patients with Multicentric Castleman's disease (MCD). The roles of interleukin-6 (IL-6) in KS and MCD have long been suspected; IL-6 promotes the growth of KS cells in culture, and IL-6 is present at elevated levels in KS and MCD lesions. Recent reports have also implicated human IL-6 (hIL-6) in the growth of HHV-8 latently infected PEL cells in soft agar and in inoculated nude mice, and hIL-6 and viral IL-6 (vIL-6) enhance tumor formation in mice (1, 2, 9). vIL-6 has been shown to enhance the growth of PEL cells in culture, although IL-10 and other factors also play a role, and to be a mitogenic factor for human T-cell and murine hybridoma cell lines (5, 13, 20, 23). Since vIL-6 has been shown to be produced by uninduced PEL cell lines, in freshly isolated PEL tissue, and in MCD and at least some KS lesions, it is possible that vIL-6, in addition to hIL-6, can play a role in the development and progression of these diseases (6, 20, 23, 26, 34). In fact, because vIL-6 signaling is not restricted to cells expressing IL-6R (also called gp80, the α-subunit of the IL-6 receptor), which is required for hIL-6 signaling through the gp130 signal transducer, it is possible that vIL-6 plays a disproportionate or distinct role in the disease process (19, 22, 24, 35). Over the past few years, extensive work has been undertaken to characterize IL-6 and its receptor subunits, IL-6R and gp130. These studies include mutagenesis of ligand and receptors and their use in binding and functional experiments to try to elucidate ligand-receptor contact sites and the nature of the functional receptor-ligand complex (see, e.g., references 3, 7, 8, 11, 14, 27, 28, 29, 30, and 37). More recently, the structures of hIL-6 and the “cytokine receptor homology domain” (CHD) of human gp130 (hgp130) have been elucidated through X-ray crystallography (4, 33). Taken together, this large body of data has provided a fairly detailed picture of how the ligand and receptor subunits interact to form a complex that then mediates, through phosphorylation of cytoplasmic tyrosine residues of gp130, signal transduction via the Jak/STAT and mitogen-activated protein kinase pathways. Dimerization of gp130, the centrally important event that initiates signal transduction, occurs on formation of a hexameric complex between two molecules of IL-6 and two molecules of each of the receptor subunits (36). IL-6 can bind to IL-6R in the absence of gp130; binding to gp130 requires prior binding of IL-6 to IL-6R. Three receptor-binding regions (sites I, II, and III) have been identified; site I interacts with IL-6R, whereas sites II and III are involved in IL-6 interactions with gp130 (17, 25, 32, 33) (Fig. ​(Fig.1).1). Site II interacts with the CHD region of gp130, while site III interacts with the more proximal immunoglobulin homology domain. For IL-6R, several residues have been shown to be important for IL-6 interaction and/or functional complex formation. Some of these residues are conserved in gp130 (which is related structurally to IL-6R) (Fig. ​(Fig.1),1), and resolution of the structure of the CHD region of gp130 has shown that some occur in two interstrand loops (E-F and B2-C2) that are positioned appropriately for interactions with ligand (via site II of hIL-6) (4). We have shown previously that alteration of three adjacent residues within one of these loops (E-F) in the gp130 variant gp130.PM5 abrogates IL-6R-independent signaling by vIL-6 (35). FIG. 1 Diagrammatic representations of the cytokine receptor homology and immunoglobulin-like domains (CHD and Ig, respectively) of gp130 and IL-6R, their interactions with hIL-6, and complex formation resulting from these interactions. The figure on the left ... In this report, we have used in vitro ligand-receptor binding assays to demonstrate binding of vIL-6 to gp130 and IL-6R both independently and as a complex, and we have measured the signaling activities of our previously reported panel of vIL-6 mutants (35) in IL-6R-independent and IL-6R-dependent assays (employing transfected gp130 or gp130.PM5 plus IL-6R) to identify residues within vIL-6 that potentially are important for interactions with gp130 and/or IL-6R. Two variants that were found to be defective in signaling through gp130 alone were able to abrogate native vIL-6 activity, indicating competitive interactions with receptor. Our data demonstrate that vIL-6 is indeed able to bind directly and independently to IL-6R and gp130 and that vIL-6 can induce IL-6R–gp130 complexing, and we identify vIL-6 residues that are important for vIL-6–gp130 and vIL-6–gp130–IL-6R functional complex formation.