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Characterization of Murine Cytomegalovirus m157 from Infected Cells and Identification of Critical Residues Mediating Recognition by the NK Cell Receptor Ly49H¹

Department of Pathology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242

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 m157 glycoprotein of murine CMV (MCMV). The Ly49H-m157 interaction underlies a potent resistance mechanism (Cmv1) 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 m157 in isolation and for MCMV-infected cells, we show that m157 is expressed in multiple isoforms with marked differences in abundance between infected fibroblasts (high) and macrophages (low). At the cell surface, m157 is exclusively a glycosylphosphatidylinositol-associated protein in MCMV-infected cells. Through random and site-directed mutagenesis of m157, we identify unique residues that provide for efficient cell surface expression of m157 but fail to activate Ly49H-expressing reporter cells. These m157 mutations are predicted to alter the conformation of a putative m157 interface with Ly49H, one that relies on the position of a critical 0 helix of m157. These findings support an emerging model for a novel interaction between this important NK cell receptor and its viral ligand.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported in part by National Institutes of Health R56 AI063226-01.

² Address correspondence and reprint requests to Dr. Jonathan Heusel, Department of Pathology/1030 Medical Laboratories, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242. E-mail address: jon-heusel{at}uiowa.edu

³ Abbreviations used in this paper: MCMV, murine cytomegalovirus; β₂m, β₂-microglobulin; CPRG, chlorophenol red-β-D-galactopyranoside; GPI, glycosylphosphatidylinositol; GPI-AP, GPI-associated protein; HSA, heat-stable Ag; IP, immunoprecipitation; MOI, multiplicity of infection; PI-PLC, phosphatidylinositol-specific phospholipase C; wt, wild type.