Abstract
Platelet-derived growth factor receptor alpha (PDGFRα) serves as an entry receptor for the human cytomegalovirus (HCMV), and soluble PDGFRα-Fc can neutralize HCMV at a half-maximal effective concentration (EC50) of about 10 ng/ml. While this indicates a potential for usage as an HCMV entry inhibitor PDGFRα-Fc can also bind the physiological ligands of PDGFRα (PDGFs), which likely interferes with the respective signaling pathways and represents a potential source of side effects. Therefore, we tested the hypothesis that interference with PDGF signaling can be prevented by mutations in PDGFRα-Fc or combinations thereof, without losing the inhibitory potential for HCMV. To this aim, a targeted mutagenesis approach was chosen. The mutations were quantitatively tested in biological assays for interference with PDGF-dependent signaling as well as inhibition of HCMV infection and biochemically for reduced affinity to PDGF-BB, facilitating quantification of PDGFRα-Fc selectivity for HCMV inhibition. Mutation of Ile 139 to Glu and Tyr 206 to Ser strongly reduced the affinity for PDGF-BB and hence interference with PDGF-dependent signaling. Inhibition of HCMV infection was less affected, thus increasing the selectivity by factor 4 and 8, respectively. Surprisingly, the combination of these mutations had an additive effect on binding of PDGF-BB but not on inhibition of HCMV, resulting in a synergistic 260fold increase of selectivity. In addition, a recently reported mutation, Val 242 to Lys, was included in the analysis. PDGFRα-Fc with this mutation was fully effective at blocking HCMV entry and had a drastically reduced affinity for PDGF-BB. Combining Val 242 to Lys with Ile 139 to Glu and/or Tyr 206 to Ser further reduced PDGF ligand binding beyond detection. In conclusion, this targeted mutagenesis approach identified combinations of mutations in PDGFRα-Fc that prevent interference with PDGF-BB but maintain inhibition of HCMV, which qualifies such mutants as candidates for the development of HCMV entry inhibitors.
Highlights
Human cytomegalovirus (HCMV) is a ubiquitous pathogen that is found worldwide in 45– 100% of the population [1]
PDGFRα-Fc binds to the virus and blocks infection, but it is more potent and has a broader activity of inhibition which makes it a promising alternative
A problem is that PDGFRα-Fc can bind to the virus and to platelet-derived growth factors (PDGFs) which are important growth factors involved in cell-cycle regulation and tissue development
Summary
Human cytomegalovirus (HCMV) is a ubiquitous pathogen that is found worldwide in 45– 100% of the population [1]. With a half-maximal effective concentration (EC50) of 10 ng/ml in fibroblasts, inhibition of fibroblast infection by PDGFRα-Fc is highly efficient as compared to neutralization by monoclonal antibodies [29,32,33,34] This decoy receptor binds to gH/gL/gO on the Sequence modification of PDGFRα-Fc for usage as an HCMV entry inhibitor virus particles and prevents them from binding to and penetrating into host cells [29]. The efficient neutralization across different cell-types sets PDGFRα-Fc apart from many of the monoclonal antibodies that have been clinically tested because they are directed against components of the viral pentamer complex and inhibit infection of endothelial and epithelial cells more efficiently than infection of fibroblasts [32,35,36,37]
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