Influence of Human Cytomegalovirus Glycoprotein O Polymorphism on the Inhibitory Effect of Soluble Forms of Trimer- and Pentamer-Specific Entry Receptors

Abstract

Human cytomegalovirus (HCMV) envelope glycoprotein complexes, gH/gL/gO-trimer and gH/gL/UL128L-pentamer, are important for cell-free HCMV entry. While soluble Nrp2-Fc (sNrp2-Fc) interferes with epithelial/endothelial cell entry through UL128, soluble PDGFRα-Fc (sPDGFRα-Fc) interacts with gO thereby inhibiting infection of all cell types. Since gO is the most variable subunit we investigated the influence of gO polymorphism on the inhibitory capacities of sPDGFRα-Fc and sNRP2-Fc. Accordingly, gO genotype 1c (GT1c) sequence was fully or partially replaced by gO GT2b, GT3, GT5 sequences in TB40-BAC4-luc background. All mutants were tested for fibroblast and epithelial cell infectivity, for virions' gB, gH, and gO content, and for infection inhibition by sPDGFRα-Fc and sNrp2-Fc. Full-length and partial gO GT swapping may increase epithelial-to-fibroblast ratios due to subtle alterations in fibroblast and/or epithelial infectivity but without substantial changes in mutant virions' gB and gH levels. All gO GT mutants except recombinant gO GT1c/3 displayed a near-complete inhibition at 1.25 μg/ml sPDGFRα-Fc on epithelial cells (98% versus 91%) and all a complete inhibition on fibroblasts (≥ 99%). While gO GT replacement did not influence sNrp2-Fc inhibition at 1.25 μg/ml on epithelial cells (97%-99%), it rendered recombinant mutant GT1c/3 moderately accessible to fibroblasts inhibition (40%). In contrast to the steep sPDGFRα-Fc inhibition curves (slope >1.0), sNrp2-Fc dose-response curves on epithelial cells displayed slopes of ∼1.0 suggesting functional differences between these entry inhibitors. Our findings demonstrate that artificially generated gO recombinants rather than the major gO genotypic forms may affect the inhibitory capacities of sPDGFRα and sNRP2, respectively, in a cell-dependent manner. IMPORTANCE Human cytomegalovirus (HCMV) is known for its broad cell tropism as reflected by the different organs and tissues affected by HCMV infection. Hence, inhibition of HCMV entry into distinct cell types could be considered as a promising therapeutic option to limit cell-free HCMV infection. Soluble forms of cellular entry receptor PDGFRα rather than those of entry receptor neuropilin-2 inhibit infection of multiple cell types. sPDGFRα specifically interacts with gO of the trimeric gH/gL/gO envelope glycoprotein complex. HCMV strains may differ with respect to the virions' amount of trimer and the highly polymorphic gO sequence. In this study, we show that the major gO genotypes of HCMV that are also found in vivo are similarly well inhibited by sPDGFRα. Novel gO genotypic forms potentially emerging through recombination, however, may evade sPDGFRα inhibition on epithelial cells. These findings provide useful additional information when anti-HCMV therapeutic compounds based on sPDGFRα will be developed.