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

Abstract

A trimeric glycoprotein complex on the surface of human cytomegalovirus (HCMV) binds to platelet-derived growth factor (PDGF) receptor α (PDGFRα) to mediate host cell recognition and fusion of the viral and cellular membranes. Soluble PDGFRα 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, PDGFRα 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 PDGFRα 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 PDGFRα 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.

Targeted mutagenesis on PDGFRα-Fc identifies amino acid modifications that allow efficient inhibition of HCMV infection while abolishing PDGF sequestration

“…Like with the previous PDGFRα-Fc mutants, all of them were expressed as efficient as wild type and did not show any signs of degradation or aggregation (S1 Table and S5 Fig) . After purification, these mutants were first tested regarding their ability to inhibit HCMV infection. In agreement with the data presented in Park et al [50], the V242K mutation did not affect HCMV inhibition (Fig 9). Introduction of V242K into PDGFRα-Fc I139E or Y206S did also not significantly alter the efficiency of HCMV inhibition.…”

“…To identify amino acid exchanges that would reduce PDGF sequestration the most and HCMV inhibition the least, hypothesis driven mutations at positions 139 and 206 were introduced into PDGFRα-Fc. We assumed that PDGF binding should be disrupted by changing either size or charge of respective side chains, since the strong affinity of PDGFRα to its PDGF ligands seems to involve hydrophobic interactions paired with an electrostatic network of hydrogen bonds [ 47 , 50 , 51 ]. However, whether those mutations would differentially affect PDGF binding and HCMV neutralization was not predictable.…”

“…In a comprehensive screening approach recently described by Park et al [ 50 ] the mutations Y206S and V242K were found most promising for abrogation of PDGF binding. As our previous experiments had demonstrated that combination of Y206S with I139E leads to strong reduction in PDGF binding affinity, the question was if combinations including V242K would further add to this effect.…”

“…A recently published study by Park et al [ 50 ] identified Tyr 206 and additionally Val 242 as important sites for PDGF binding that can be mutated without loss of efficiency for HCMV inhibition. Park et al [ 50 ] performed a comprehensive mutation of all positions in domains 2 and 3 of PDGFRα and screened in a high-throughput flow cytometry-based competition assay for binding of HCMV and PDGFs. In contrast, we tested deletion mutants for compatibility with HCMV inhibition, followed by a hypothesis-driven selection of site-specific mutations.…”

ACE2-Based Decoy Receptors for SARS Coronavirus 2

“…The assumption is that administration of a catalytically active decoy receptor will adversely interact with physiology to have unacceptable toxicity. Soluble decoy receptors for other viruses have also been engineered to eliminate the normal biological activity for safety and efficacy reasons 66 . However, there are strong arguments that ACE2 catalytic activity, even if elevated, might be beneficial for treating COVID-19 symptoms.…”