Principles of viral immune evasion elucidated by biophysical studies of a herpesvirus-encoded chemokine decoy receptor

Large DNA viruses have developed immune evasion strategies to undermine host anti-viral defenses, and given that chemokines are essential mediators of immune homeostasis and pathogen surveillance, this network is a favored target. One tactic employed by viruses is secretion of high-affinity binding proteins that act as chemokine scavengers, exemplified by M3 from murine gammaherpesvirus68. The goal of this dissertation is to understand how the M3 decoy receptor broadly neutralizes chemokine activity.; To investigate the structural basis of chemokine sequestration by M3, the structure of M3 was determined alone and in complex with the chemokine MCP-1. M3 is a novel two-domain beta-sandwich protein forming an antiparallel dimer that binds MCP-1 with a 2:2 stoichiometry. This study revealed that M3 structurally mimics GPCRs to sequester chemokines by a competitive inhibition mechanism.; To address the hypothesis that M3 uses conformational plasticity and electrostatic complementarity to promiscuously bind chemokines, crystallographic analysis was extended to all four classes of chemokines, including lymphotactin, fractalkine, IP-10 and IL-8. These studies were complemented by surface plasmon resonance (SPR)-derived affinity and kinetic measurements, which demonstrated that chemokines rapidly associate with M3 in part due to favorable electrostatic interactions. Structure analysis further suggested that M3 competitively inhibits chemokine-glycosaminoglyans (GAG) interactions. SPR and cellular competition assays established that M3 stoichiometrically inhibits chemokine binding to cell-surface GAGs. Furthermore, structural comparison revealed that conformational plasticity of M3 facilitates the coordinated GPCR and GAG blockade that disrupts interactions with infiltrating leukocytes and the formation of chemotactic gradients.; M3 broadly sequesters CC, C and CX3C chemokines, yet is selective within the CXC class. To examine selectivity, structure-based mutagenesis and kinetic assays were used to identify residues that contribute to extended M3 off-rates. This analysis revealed that M3 is optimized for certain ligands and showed how kinetic stability can be improved for chemokines with short half-lives. Methodology has also been developed to create M3 variants with altered binding properties using yeast surface display, with the goal of developing chemokine-specific reagents that may be exploited as anti-inflammatory therapeutics. Studies to test the efficacy of M3 variants in the murine model of Multiple Sclerosis (EAE) have been initiated.