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

Abstract

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.

Peptide Derivatives of Platelet-Derived Growth Factor Receptor Alpha Inhibit Cell-Associated Spread of Human Cytomegalovirus

“…As the same glycoproteins that mediate cell-free infection of HCMV are also involved in cell-associated spread [ 35 ], it was tempting to speculate that this transmission mode can be targeted by entry inhibitors. This assumption is supported by our finding that certain peptides derived from the extracellular domain of the HCMV receptor PDGFRα, which block the adsorption and penetration of cell-free virions [ 48 ], also inhibit the cell-associated spread of clinical HCMV isolates and the cell-associated model virus Merlin. This effect was mediated by reducing the number and the penetration efficiency of transferred viral particles, thus resembling the effect on the cell-free virus.…”

“…In line with this assumption, GD30 reduced the number of particles that were transferred from late stage infected cells to their uninfected neighbors, and the majority of these particles were not penetrated. This resembles the effect of PDGFRα-Fc and its peptide derivatives on the adsorption and penetration of free virions [ 18 , 48 ]. The inhibition of cell-free infection by GD30 was HCMV-specific and depended on the expression of gO, which further supports the idea that peptides and full-length proteins share the same mode of action.…”

“…Regarding the mode of action of GD30 in the inhibition of cell-associated spread of HCMV, it appeared reasonable to assume that the peptide inhibits the transfer of infectious viral particles. Regarding the effect on cell-free virions, we had previously found that PDGFRα and its derived peptides inhibit the adsorption and penetration of viral particles [ 18 , 48 ]. By analogy, one could hypothesize that the PDGFRα derivative GD30 decreases the number of particles that can be transferred from productively infected cells to neighboring cells during cell-associated spread and/or their penetration efficiency.…”

“…The inhibitory effect of the decoy receptor PDGFRα-Fc is based on its interaction with the gO component of the trimeric gH/gL/gO complex [ 17 , 18 , 19 ], and the selection of resistance-conferring mutations indicated that the peptide derivative GT40 also targets the trimer [ 48 ], which is also plausible considering the structural information on the interaction of the GT40-peptide with gO [ 49 , 50 ]. Since GD30 is merely a truncated version of GT40, interaction with gO is also conceivable as a molecular mechanism of this peptide.…”

Viral and Cellular Factors Contributing to the Hematogenous Dissemination of Human Cytomegalovirus via Polymorphonuclear Leukocytes

“…Thus, a sufficient number of recipient endothelial cells were infected to allow the detection of stronger inhibitory effects that would be caused by the entry inhibitors. HCMV-specific hyperimmunoglobulin (Cytotect, CP Biotest, Dreieich, Germany), recombinant PDGFRα-Fc and Nrp2-Fc (R&D Systems), and the PDGFRα-derived 30- and 40-mer peptides GD30, GT40 and IK40 [ 20 , 33 , 52 ] (Phtdpeptides, Shanghai, China) were added during incubation of either anti-integrin β2-treated or untreated PMNs with recipient HEC-LTTs at a concentration to achieve complete inhibition of cell-free virus (at least 10 × EC50). This resulted in concentrations of 0.5 mg/mL for hyperimmunoglobulin, 120 ng/mL for PDGFRα-Fc and 590 ng/mL for Nrp2-Fc.…”