Abstract
Glycoprotein O (gO) of the human cytomegalovirus (HCMV) is the critical subunit of the envelope trimer gH/gL/gO as it interacts with platelet-derived growth factor alpha receptor upon fibroblast entry, and triggers gB-mediated fusion for fibroblast and epithelial cell infection. Eight genotypes (GT) of the highly polymorphic gO gene are described, yet it is unclear whether the distinct GTs differ in their function. Thus, we aimed to elucidate potential functional differences between two highly diverse gO GTs in an otherwise genomically identical HCMV strain. Therefore, resident gO GT1c sequence of strain TB40-BAC4-luc was entirely replaced by gO GT4 of strain Towne and both, GT1c and GT4 viruses, were investigated for their growth properties in fibroblasts and epithelial cells. In addition, two conserved gO cysteines involved in gH/gL/gO stabilization were mutated to serine either in GT1c (C218S and C343S) or GT4 (C216S and C336S) and their effects on cell-free infectivity were assessed. GT4 viruses displayed a significantly enhanced epithelial cell tropism and this resulted in higher virus release upon replication in epithelial cells when compared to GT1c viruses. Further, when the two cysteines were individually mutated in gO GT1c no impairment in cell-free infectivity was observed. This, however, was in sharp contrast to gO GT4, in which both of the corresponding cysteine mutations led to a substantial reduction in cell-free infectivity which was even more pronounced upon mutation of GT4-C336 than of GT4-C216. In conclusion, these findings provide evidence that the two highly diverse gO genotypes, GT1c and GT4, differ in their functional properties as revealed by their different infection capacities for epithelial cells and by their different responsiveness to mutation of strictly conserved cysteine residues. Thus, it is likely that the gO heterogeneity influences cell-free infectivity of HCMV also in vivo which may have important implications for virus host transmission.
Highlights
Human cytomegalovirus (HCMV) is a ubiquitously distributed human betaherpesvirus with 40–90% of adults infected worldwide
Recombination-positive E. coli were subjected to kanamycin selection, and the introduced non-HCMV sequences were removed within E. coli by cleavage at the I-Sce I site and a second red recombination
Whole HCMV genome sequencing of both Glycoprotein O (gO) GT1c (GT1c_1 and GT1c_2) and gO GT4 mutant virus stocks (GT4_1 and GT4_2) confirmed that no additional mutations emerged during reconstitution
Summary
Human cytomegalovirus (HCMV) is a ubiquitously distributed human betaherpesvirus with 40–90% of adults infected worldwide. Human cytomegalovirus has a large dsDNA genome with about 236 kb coding for ∼165 genes (Dolan et al, 2004; Sijmons et al, 2014). The current model proposes that the trimer is necessary for entry in all cell types while the pentamer is required for entry into epithelial and endothelial cells (Jiang et al, 2008; Wille et al, 2010; Kabanova et al, 2016), and it is further suggested that the relative amounts of both complexes define the cell tropism of virus progenies (Murrell et al, 2013; Zhou et al, 2013, 2015; Laib Sampaio et al, 2016)
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