Characterization of murine cytomegalovirus ml57 from infected cells and identification of critical residues mediating recognition by the NK cell receptor Ly49H

Activated NK cells mediate potent cytolytic and secretory effector functions and are vital components of the early antiviral immune response. NK cell activities are regulated by the assortment of inhibitory receptors that recognize MHC class I ligands expressed on healthy cells and activating receptors that recognize inducible host ligands or ligands that are not well characterized. The activating Ly49H receptor of mouse NK cells is unique in that it specifically recognizes a virally encoded ligand, the ml57 glycoprotein of murine CMV (MCMV). The Ly49H-ml57 interaction underlies a potent resistance mechanism (Cmvl) in C57BL/6 mice and serves as an excellent model in which to understand how NK cells are specifically activated in vivo, as similar receptor systems are operative for human NK cells. For transduced cells expressing ml57 in isolation and for MCMV-infected cells, we show that ml57 is expressed in multiple isoforms with marked differences in abundance between infected fibroblasts (high) and macrophages (low). At the cell surface, ml57 is exclusively a glycosylphosphatidylinositol-associated protein in MCMV-infected cells. Through random and site-directed mutagenesis of ml57, we identify unique residues that provide for efficient cell surface expression of ml57 but fail to activate Ly49H-expressing reporter cells. These ml57 mutations are predicted to alter the conformation of a putative ml57 interface with Ly49H, one that relies on the position of a critical-α helix of ml57. These findings support an emerging model for a novel interaction between this important NK cell receptor and its viral ligand.