Abstract
NKG2D is a crucial Natural Killer (NK) cell activating receptor, and the murine cytomegalovirus (MCMV) employs multiple immunoevasins in order to avoid NKG2D-mediated activation. One of the MCMV immunoevasins, gp40 (m152), downregulates the cell surface NKG2D ligand, RAE-1γ, thus limiting NK cell activation. This study establishes the molecular mechanism by which gp40 retains RAE-1γ in the secretory pathway. Using flow cytometry and pulse chase analysis, we demonstrate that gp40 retains RAE-1γ in the early secretory pathway, and that this effect depends on the binding of gp40 to a host protein, TMED10, a member of the p24 protein family. We also show that the TMED10-based retention mechanism can be saturated, and that gp40 has a backup mechanism as it masks RAE-1γ on the cell surface, blocking the interaction with the NKG2D receptor and thus NK cell activation.
Summary statement MCMV immunoevasin gp40 inhibits the NKG2D-activating ligand RAE-1γ by intracellular retention that depends on the p24 member TMED10, and additionally by masking it at the cell surface.
List of Symbols and Abbreviations
- HCMV
- Human Cytomegalovirus
- MCMV
- murine cytomegalovirus
- NK cell
- natural killer cell
- Endo F1
- endoglycosidase F1
- TMED10
- transmembrane p24 trafficking protein 10
- CX1
- monoclonal anti-RAE 1γ antibody
- gp40-
- cells transfected with HA-RAE-1γ without gp40
- gp40+
- cells transfected with HA-RAE-1γ and gp40
- gp40LM
- gp40 linker mutant
- gp40WT
- gp40 wild type
- PDM
- Protein Deglycosylation Mix
- APC
- APC-conjugated secondary antibody
- RE-IP
- re-immunoprecipitation.
