The N Terminus of Human Cytomegalovirus Glycoprotein O Is Important for Binding to the Cellular Receptor PDGFRα

Abstract

The human cytomegalovirus (HCMV) glycoprotein complex gH/gL/gO is required for the infection of cells by cell-free virions. It was recently shown that entry into fibroblasts depends on the interaction of gO with the platelet-derived growth factor receptor alpha (PDGFR␣). This interaction can be blocked with soluble PDGFR␣-Fc, which binds to HCMV virions and inhibits entry. The aim of this study was to identify parts of gO that contribute to PDGFR␣ binding. In a systematic mutational approach, we targeted potential interaction sites by exchanging conserved clusters of charged amino acids of gO with alanines. To screen for impaired interaction with PDGFR␣, virus mutants were tested for sensitivity to inhibition by soluble PDGFR␣-Fc. Two mutants with mutations within the N terminus of gO (amino acids 56 to 61 and 117 to 121) were partially resistant to neutralization. To validate whether these mutations impair interaction with PDGFR␣-Fc, we compared binding of PDGFR␣-Fc to mutant and wild-type virions via quantitative immunofluorescence analysis. PDGFR␣-Fc staining intensities were reduced by 30% to 60% with mutant virus particles compared to wild-type particles. In concordance with the reduced binding to the soluble receptor, virus penetration into fibroblasts, which relies on binding to the cellular PDGFR␣, was also reduced. In contrast, PDGFR␣-independent penetration into endothelial cells was unaltered, demonstrating that the phenotypes of the gO mutant viruses were specific for the interaction with PDGFR␣. In conclusion, the mutational screening of gO revealed that the N terminus of gO contributes to efficient spread in fibroblasts by promoting the interaction of virions with its cellular receptor. IMPORTANCE The human cytomegalovirus is a highly prevalent pathogen that can cause severe disease in immunocompromised hosts. Currently used drugs successfully target the viral replication within the host cell, but their use is restricted due to side effects and the development of resistance. An alternative approach is the inhibition of virus entry, for which understanding the details of the initial virus-cell interaction is desirable. As binding of the viral gH/gL/gO complex to the cellular PDGFR␣ drives infection of fibroblasts, this is a potential target for inhibition of infection. Our mutational mapping approach suggests the N terminus as the receptor binding portion of the protein. The respective mutants were partially resistant to inhibition by PDGFR␣-Fc but also attenuated for infection of fibroblasts, indicating that such mutations have little if any benefit for the virus. These findings highlight the potential of targeting the interaction of gH/gL/gO with PDGFR␣ for therapeutic inhibition of HCMV.

Influence of human cytomegalovirus glycoprotein O polymorphism on the inhibitory effect of soluble forms of trimer- and pentamer-specific entry receptors

“…Although this peptide site overlaps with the recombination site of GT1c/GT3, the specific sequence remained unchanged upon recombination suggesting that sPDGFRα binding to this recombinant form of gO is not impaired. This presumption fits well to the finding that gO GT1c/GT3 mutant does not display a phenotype in fibroblast infectivity while mutants with a mutation in this particular peptide sequence showed reduced penetration into fibroblasts (31). Hence, we assume that the impaired sPDGFRα inhibition for gO GT1c/GT3 mutant on epithelial cells is not caused by a lower binding of sPDGFRα to gO but rather by an impaired interference with a downstream entry step mediated by the trimer.…”

“…Remarkably, one of the two recombinant mutants, gO GT1c/GT3, displayed a significantly lower sensitivity for sPDGFRα inhibition on epithelial cells than the other mutants while the fibroblast inhibition was similarly effective. As mentioned above this mutant comprises its recombination site in the highly polymorphic N-terminal region of the protein, which only recently was suggested to contain the PDGFRα receptor binding domain (31). By mutational analysis the authors identified a small stretch from amino acid 117 to 121 causing the strongest impairment of sPDGFRα binding to virus particles and consequently also a reduced virus penetration into fibroblasts.…”

“…Both PDGFRα and NRP2 likely function as the primary entry receptors for the trimer and pentamer, respectively, however, the modes of entry downstream of receptor binding may substantially differ. In particular, it appears that the trimer functions at steps which are required for entry into all cell types (14) which makes sPDGFRα or derivatives thereof a promising therapeutic tool against HCMV (30, 31). In the present study, we now aimed to assess how gO polymorphism influences the inhibitory capacity of sPDGFRα and sNRP2, respectively.…”

Convalescent Immunity to Guinea Pig Cytomegalovirus Induces Limited Cross Strain Protection against Re-Infection but High-Level Protection against Congenital Disease

“…Similar to HCMV gO, GP74 protein (22122 strain) is heavily glycosylated with 13 predicted sites [ 15 ]. In the TAMYC strain, the first two predicted N-glycosylation sites (N-X-T/A) are lost (amino acids 41 and 57) [ 15 ] ( Figure 1 ) and the potential impact of this loss in this N-terminal domain glycosylation awaits further study but the second glycosylation site is conserved between HCMV and GPCMV gO [ 42 ]. Potentially modified glycosylation could impact on ability of gO to interact with PDGFRA cell receptor [ 30 ].…”

Engineered receptors for human cytomegalovirus that are orthogonal to normal human biology

“…This concern is partially alleviated by the observation that the engineered PDGFRα mutants maintain high binding to soluble HCMV trimer from the Merlin clinical strain [58] ( Fig 3C), which has relatively high natural sequence variation compared to TB40/E (79% identity between glycoproteins O). Variation is particularly high in the N-terminus of glycoprotein O where important interactions to PDGFRα reside [54].…”