Targeted mutagenesis on PDGFRα-Fc identifies amino acid modifications that allow efficient inhibition of HCMV infection while abolishing PDGF sequestration.
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...
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Public Library of Science (PLoS)
2021-03-01
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| Series: | PLoS Pathogens |
| Online Access: | https://journals.plos.org/plospathogens/article/file?id=10.1371/journal.ppat.1009471&type=printable |
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| author | Svenja Feldmann Immanuel Grimm Dagmar Stöhr Chiara Antonini Peter Lischka Christian Sinzger Cora Stegmann |
| author_facet | Svenja Feldmann Immanuel Grimm Dagmar Stöhr Chiara Antonini Peter Lischka Christian Sinzger Cora Stegmann |
| author_sort | Svenja Feldmann |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-8e05b5ed7837460fa1831522177f2ed4 |
| institution | DOAJ |
| issn | 1553-7366 1553-7374 |
| language | English |
| publishDate | 2021-03-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS Pathogens |
| spelling | doaj-art-8e05b5ed7837460fa1831522177f2ed42025-08-20T02:41:11ZengPublic Library of Science (PLoS)PLoS Pathogens1553-73661553-73742021-03-01173e100947110.1371/journal.ppat.1009471Targeted mutagenesis on PDGFRα-Fc identifies amino acid modifications that allow efficient inhibition of HCMV infection while abolishing PDGF sequestration.Svenja FeldmannImmanuel GrimmDagmar StöhrChiara AntoniniPeter LischkaChristian SinzgerCora StegmannPlatelet-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.https://journals.plos.org/plospathogens/article/file?id=10.1371/journal.ppat.1009471&type=printable |
| spellingShingle | Svenja Feldmann Immanuel Grimm Dagmar Stöhr Chiara Antonini Peter Lischka Christian Sinzger Cora Stegmann Targeted mutagenesis on PDGFRα-Fc identifies amino acid modifications that allow efficient inhibition of HCMV infection while abolishing PDGF sequestration. PLoS Pathogens |
| title | Targeted mutagenesis on PDGFRα-Fc identifies amino acid modifications that allow efficient inhibition of HCMV infection while abolishing PDGF sequestration. |
| title_full | Targeted mutagenesis on PDGFRα-Fc identifies amino acid modifications that allow efficient inhibition of HCMV infection while abolishing PDGF sequestration. |
| title_fullStr | Targeted mutagenesis on PDGFRα-Fc identifies amino acid modifications that allow efficient inhibition of HCMV infection while abolishing PDGF sequestration. |
| title_full_unstemmed | Targeted mutagenesis on PDGFRα-Fc identifies amino acid modifications that allow efficient inhibition of HCMV infection while abolishing PDGF sequestration. |
| title_short | Targeted mutagenesis on PDGFRα-Fc identifies amino acid modifications that allow efficient inhibition of HCMV infection while abolishing PDGF sequestration. |
| title_sort | targeted mutagenesis on pdgfrα fc identifies amino acid modifications that allow efficient inhibition of hcmv infection while abolishing pdgf sequestration |
| url | https://journals.plos.org/plospathogens/article/file?id=10.1371/journal.ppat.1009471&type=printable |
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