Engineered receptors for human cytomegalovirus that are orthogonal to normal human biology

A trimeric glycoprotein complex on the surface of human cytomegalovirus (HCMV) binds to platelet-derived growth factor (PDGF) receptor alpha (PDGFR alpha) to mediate host cell recognition and fusion of the viral and cellular membranes. Soluble PDGFR alpha potently neutralizes HCMV in tissue culture, and its potential use as an antiviral therapeutic has the benefit that any escape mutants will likely be attenuated. However, PDGFR alpha binds multiple PDGF ligands in the human body as part of developmental programs in embryogenesis and continuing through adulthood. Any therapies with soluble receptor therefore come with serious efficacy and safety concerns, especially for the treatment of congenital HCMV. Soluble virus receptors that are orthogonal to human biology might resolve these concerns. This engineering problem is solved by deep mutational scanning on the D2-D3 domains of PDGFR alpha to identify variants that maintain interactions with the HCMV glycoprotein trimer in the presence of competing PDGF ligands. Competition by PDGFs is conformation-dependent, whereas HCMV trimer binding is independent of proper D2-D3 conformation, and many mutations at the receptor-PDGF interface are suitable for functionally separating trimer from PDGF interactions. Purified soluble PDGFR alpha carrying a targeted mutation succeeded in displaying wild type affinity for HCMV trimer with a simultaneous loss of PDGF binding, and neutralizes trimer-only and trimer/pentamer-expressing HCMV strains infecting fibroblasts or epithelial cells. Overall, this work makes important progress in the realization of soluble HCMV receptors for clinical application. Author summary Human cytomegalovirus (HCMV) causes severe disease in transplant recipients and immunocompromised patients, and infections in a fetus or neonate are responsible for life-long neurological defects. Cell entry is in part mediated by a trimeric glycoprotein complex on the viral surface, which binds tightly to the host receptor PDGFR alpha. The soluble extracellular region of PDGFR alpha can be used as an antiviral agent to potently neutralize the virusin vitro. However, PDGFR alpha ordinarily binds growth factors in the human body to regulate developmental programs, which will limit thein vivoefficacy and safety of soluble PDGFR alpha. Using saturation mutagenesis and selections in human cell culture, mutations in PDGFR alpha are identified that eliminate off-target growth factor interactions while preserving HCMV binding and neutralization.