Selection of Human Cytomegalovirus Mutants with Resistance against PDGFRα-Derived Entry Inhibitors

The human cytomegalovirus (HCMV) infects fibroblasts via an interaction of its envelope glycoprotein gO with the cellular platelet-derived growth factor receptor alpha (PDGFRα), and soluble derivatives of this receptor can inhibit viral entry. We aimed to select mutants with resistance against PDGFRα-Fc and the PDGFRα-derived peptides GT40 and IK40 to gain insight into the underlying mechanisms and determine the genetic barrier to resistance. An error-prone variant of strain AD169 was propagated in the presence of inhibitors, cell cultures were monitored weekly for signs of increased viral growth, and selected viruses were tested regarding their sensitivity to the inhibitor. Resistant virus was analyzed by DNA sequencing, candidate mutations were transferred into AD169 clone pHB5 by seamless mutagenesis, and reconstituted virus was again tested for loss of sensitivity by dose-response analyses. An S48Y mutation in gO was identified that conferred a three-fold loss of sensitivity against PDGFRα-Fc, a combination of mutations in gO, gH, gB and gN reduced sensitivity to GT40 by factor 4, and no loss of sensitivity occurred with IK40. The resistance-conferring mutations support the notion that PDGFRα-Fc and GT40 perturb the interaction of gO with its receptor, but the relatively weak effect indicates a high genetic barrier to resistance.