Monocyte chemoattractant protein-1 (MCP-1) is a chemotactic cytokine mainly acting on monocytes and T cells that elicits its biological effects by interacting with the seven-transmembrane helix receptor CCR2B. The vaccinia virus strain Lister and many other poxviruses express soluble proteins (vCCI) that bind MCP-1 and other CC chemokines and inhibit their function. In order to define the interaction site of MCP-1 with vCCI from vaccinia, surface exposed residues of MCP-1 were identified and mutated to alanine. The MCP-1 variants were expressed, purified, and their interaction with vCCI was characterized. The site on MCP-1 for vCCI binding is dominated by arginine 18 with important additional contributions from tyrosine 13 and arginine 24. These residues define a binding site that largely overlaps with the CCR2B receptor interaction site. The viral chemokine-binding protein vCCI thus inhibits the biological function of MCP-1 by directly masking its CCR2B receptor-binding site.Chemokines are small (8 -14 kDa) structurally related proteins that regulate cell trafficking of various leukocyte subtypes through interaction with a set of G protein-coupled receptors. The two major subfamilies are the CXC chemokines which act on neutrophils and non-hemopoietic cells and the CC chemokines which bind to receptors mainly expressed on monocytes, T cells, eosinophils, and basophils. Additional members of the chemokine family are the C chemokine lymphotactin and the CX3C chemokine fraktalkine. Chemokines are important for the development, homeostasis, and function of the immune system and play a pivotal role in host defense (for reviews, see Refs. 1-3).Given the importance of chemokines for defense against pathogens it is no surprise that viruses themselves developed mechanisms to neutralize the function of chemokines to further their own propagation or evade host defense (4). Poxviruses express secreted chemokine neutralizing proteins termed virus-encoded chemokine-binding proteins (vCKBP).1 Two families of vCKBPs have thus far been identified that use distinct mechanisms of chemokine neutralization (5). The vCKBP-1 family binds C, CC, and CXC chemokines with low affinity, possibly by interaction with the proteoglycan-binding site on chemokines, thereby interfering with the proper localization and presentation of chemokines in inflamed tissues (6, 7). The vCKBP-2 proteins, however, bind preferably CC chemokines with high affinity and inhibit the interaction of chemokines with their cellular receptors (8 -10). vCKBP-2 proteins have been identified in strains of vaccinia (11), cowpox (12), rabbitpox (13), myxomavirus (14), and variola virus (15). Prototypic members of this protein family are the M-T1 gene product from myxoma virus and the rabbitpox virus major secreted 35-kDa protein (10). In rabbits infected with a rabbitpox virus which had the gene for the secreted 35-kDa protein deleted, an increased leukocyte influx into virus-infected tissues was observed, confirming the role of this protein in inhibition of chemokine-medi...

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A method for the identification of high-affinity ligands to SH2 domains by fluorescence-activated bead sorting (FABS) was established. Recombinant SH2 domains, expressed as glutathione S-transferase (GST) fusion proteins, were incubated with a phosphotyrosine (Y*)-containing peptide library. 6.4 ؋ 10 5 individual peptides of nine amino acids in length (EPX 6 Y*X 19 X 7 X 19 X 7 X 6) were each displayed on beads. Phosphopeptide interaction of a given SH2 domain was monitored by binding of fluorescein isothiocyanate-labeled antibodies directed against GST. High-fluorescence beads were isolated by flow cytometric sorting. Subsequent pool sequencing of the selected beads revealed a distinct pattern of phosphotyrosine-containing motifs for each individual SH2 domain: the SH2 domain of the adapter protein Grb2 predominantly selected beads with the sequence Y*ENDP, whereas the C-terminal SH2 domain of the tyrosine kinase Syk selected Y*EELD, each motif representing the most frequently found residues C-terminal to the phosphotyrosine. For deconvolution studies, soluble phosphopeptides comprising variations of the Grb2 motifs were resynthesized and analyzed by surface plasmon resonance.

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