Targeted Mutagenesis On Pdgfr Alpha-Fc Identifies Amino Acid Modifications That Allow Efficient Inhibition Of Hcmv Infection While Abolishing Pdgf Sequestration

Platelet-derived growth factor receptor alpha (PDGFR alpha) serves as an entry receptor for the human cytomegalovirus (HCMV), and soluble PDGFR alpha-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 PDGFR alpha-Fc can also bind the physiological ligands of PDGFR alpha (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 PDGFR alpha-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 PDGFR alpha-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. PDGFR alpha-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 PDGFR alpha-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.Author summaryHuman cytomegalovirus is a major cause of congenital birth defects. Yet, currently the best way to avoid cytomegalovirus disease is to prevent infection of pregnant women through hygiene measures. Once the mother is infected there is no approved treatment to block transmission to the fetus. One intensively researched option is to neutralize the virus produced by the infected mother with anti-HCMV antibodies. Yet, as the efficiency of this approach remains to be demonstrated, alternative approaches need to be considered. Similar to antibodies, PDGFR alpha-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 however is that PDGFR alpha-Fc can not only bind to the virus but also to PDGFs which are important growth factors involved in cell-cycle regulation and tissue development. The results of this study offer a solution. Combinations of mutations were identified that can be introduced in PDGFR alpha-Fc to abrogate sequestration of PDGFs. Thus, the potential side effects of PDGFR alpha-Fc can be circumvented while it remains active against HCMV. These results pave the way for development of PDGFR alpha-Fc as a promising HCMV inhibitor.