title: Direct interaction of the mouse cytomegalovirus m152/gp40 immunoevasin with RAE-1 isoforms.
abstract: Cytomegaloviruses (CMVs) are ubiquitous species-specific viruses that establish acute, persistent, and latent infections. Both human and mouse CMVs encode proteins that inhibit the activation of natural killer (NK) cells by downregulating cellular ligands for the NK cell activating receptor, NKG2D. The MCMV glycoprotein m152/gp40 downregulates the surface expression of RAE-1 to prevent NK cell control in vivo. So far, it is unclear if there is a direct interaction between m152 and RAE-1 and, if so, if m152 interacts differentially with the five identified RAE-1 isoforms, which are expressed as two groups in MCMV-susceptible or -resistant mouse strains. To address these questions, we expressed and purified the extracellular domains of RAE-1 and m152 and performed size exclusion chromatography binding assays as well as analytical ultracentrifugation and isothermal titration calorimetry to characterize these interactions quantitatively. We further evaluated the role of full-length and naturally glycosylated m152 and RAE-1 in cotransfected HEK293T cells. Our results confirmed that m152 binds RAE-1 directly, relatively tightly (K(d) < 5 microM), and with 1:1 stoichiometry. The binding is quantitatively different depending on particular RAE-1 isoforms, corresponding to the susceptibility to downregulation by m152. A PLWY motif found in RAE-1beta, although contributing to its affinity for m152, does not influence the affinity of RAE-1gamma or RAE-1delta, suggesting that other differences contribute to the RAE-1-m152 interaction. Molecular modeling of the different RAE-1 isoforms suggests a potential site for the m152 interaction.
authors.affiliation: Molecular Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-1892, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Zhi
authors.forename: Janet
authors.initials: J
authors.lastname: Mans
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Paskow
authors.forename: Patrick H
authors.initials: PH
authors.lastname: Brown
authors.forename: Peter
authors.initials: P
authors.lastname: Schuck
authors.forename: Stipan
authors.initials: S
authors.lastname: Jonjić
authors.forename: Kannan
authors.initials: K
authors.lastname: Natarajan
authors.forename: David H
authors.initials: DH
authors.lastname: Margulies
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D015536:Down-Regulation; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018146:Muromegalovirus; D055655:NK Cell Lectin-Like Receptor Subfamily K / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D055655:NK Cell Lectin-Like Receptor Subfamily K; D020033:Protein Isoforms; D014764:Viral Proteins; C104783:m152 protein, cytomegalovirus