Distinct roles of extracellular domains in the EBV-encoded BILF1 receptor for signaling and MHC class I downregulation

The Epstein-Barr virus (EBV) BILF1 gene encodes a constitutively active G protein-coupled receptor (GPCR) that downregulates major histocompatibility complex (MHC) class I and induces signaling-dependent tumorigenesis. Different BILF1 homologs display highly conserved extracellular loops (ECLs) including the conserved cysteine residues involved in disulfide bridges present in class A GPCRs (GPCR-bridge between transmembrane helix 3 (TM-3) and ECL-2) and in chemokine receptors (CKR-bridge between the N-terminus and ECL-3). In order to investigate the role of the conserved residues for the receptor functions, 25 mutants were created in the extracellular domains. Luciferase reporter assays and flow cytometry were used to investigate the G protein signaling and MHC class I downregulation, in HEK293 cells. We find that the cysteine residues involved in the GPCR-bridge are important for both signaling and MHC class I downregulation, whereas the cysteine residues in the N-terminus and ECL-3 are dispensable for signaling, but important for MHC class I downregulation. Multiple conserved residues in the extracellular regions are important for the receptor-induced MHC class I downregulation, but not for signaling, indicating distinct structural requirements for these two functions. In an engineered receptor containing a binding site for Zn+2 ions in a complex with an aromatic chelator (phenanthroline or bipyridine), a ligand-driven inhibition of both the receptor signaling and MHC class I downregulation was observed. Taken together, this suggests that distinct regions in EBV-BILF1 can be pharmacologically targeted to inhibit the signaling-mediated tumorigenesis and interfere with the MHC class I downregulation leading to improved T-cell recognition of EBV-infected cells