The Viral CC Chemokine-binding Protein vCCI Inhibits Monocyte Chemoattractant Protein-1 Activity by Masking Its CCR2B-binding Site

Abstract

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...

Solution structure of the complex between poxvirus-encoded CC chemokine inhibitor vCCI and human MIP-1β

“…In the current structure, the 48th position is approached closely by the side chains of vCCI Tyr-80 and Arg-89, so substitution of Lys-48 by Ala in MIP-1␤ could alleviate steric crowding and poor electrostatic effects of the WT protein. In support of this, the analogous mutation in MCP-1 Lys-49-Ala actually showed an increase in affinity for vCCI (27,28).…”

“…An amino acid with a shorter or a polar side chain would not be able to make these contacts and would therefore lack this component of binding affinity. Indeed, detailed mutagenesis studies of the CC chemokine MCP-1 show an Ϸ10-fold reduction in affinity when analogous residue Tyr-13 is replaced by Ala (27,28). In all human chemokines that bind tightly to vCCI, this position is an aromatic or a large hydrophobic amino acid (26), again demonstrating the importance of this residue in both the function and inhibition of CC chemokines.…”

“…3). Although this interaction is not shielded from water, mutagenesis experiments on MCP-1 indicate its significance, showing a Ͼ20-fold reduction in affinity when Arg-18 is substituted with Ala (27,28). Additionally, sequence analysis of human chemokines that bind vCCI shows a nearly universal positive charge at this position (Fig.…”

“…An extensive binding study with vaccinia vCCI using Ͼ80 chemokines from several organisms revealed 26 CC chemokines that bind with high affinity, including 13 human chemokines, such as MCP-1 (monocyte chemoattractant protein 1), MIP-1␣, MIP-1␤, and RANTES (26). To probe the chemokine residues that are important in vCCI binding, two previous studies using vCCI and variants of human CC chemokine MCP-1 suggested that the MCP-1 monomeric subunit binds vCCI through an interface that is partially overlapping with the site that MCP-1 uses to bind its natural receptors, with residues Tyr-13, Arg-18, and Arg-24 found to be particularly important (27,28). The present work displays the structural underpinning of the high affinity between poxvirus vCCI and CC chemokines.…”

Rat Aortic MCP-1 and Its Receptor CCR2 Increase With Age and Alter Vascular Smooth Muscle Cell Function

“…vCCI is a viral chemokine-binding protein expressed by the vaccinia virus strain Lister that blocks MCP-1 activity by masking the receptor binding site. 57 This molecule has been shown to ameliorate some aspects of inflammatory disease. 58 A fifth novel finding of the present study is that MCP-1 enhances migration and the ability of VSMCs from young aortas to invade a Matrigel-coated filter that mimics the extracellular matrix and that after MCP-1 treatment, these features of young VSMCs are indistinguishable from those of early-passage VSMCs from older rats.…”