Differences in Growth Properties among Two Human Cytomegalovirus Glycoprotein O Genotypes

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.

Keywords: HCMV; epithelial cells; fibroblasts; glycoprotein O; trimeric complex; tropism.

FIGURE 1

Growth curves and genome dynamics of gO GT1c and gO GT4. Fibroblasts (HFF) and epithelial cells (ARPE-19) were infected with two clones each of gO GT1c and GT4 at an MOI of 0.1 and cultured for 12 days. At the indicated time points, viral titer [TCID₅₀/ml on fibroblasts; (A,C)] and cell-free viral load [HCMV DNA copies/ml (B,D)] of cell culture supernatants were assessed. Error bars indicate SD from three replicates.

FIGURE 2

Relative epithelial cell tropism of gO GT1c and gO GT4 mutants. Fibroblasts (HFF) and epithelial cells (ARPE-19) were infected in triplicates with two clones each of gO GT1c and GT4 at low MOIs from 0.1 to 0.4 for 2 h, and incubated for further 46 h in the presence (A,B) or absence (C,D) of phosphonoacetic acid (PAA). The HFF infection capacity was set to 100% and the epithelial cell infection capacity was determined proportionally. Relative light units (RLU) were determined in triplicates. Mean values from three to six independent experiments are shown in the upper panels, error bars indicate SD. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, unpaired two-tailed t-test. In the lower panels (B,D), three single representative assays for each PAA+ and PAA– are shown.

FIGURE 3

Infectivity of single cysteine mutants for fibroblasts and epithelial cells compared to the respective parental strains gO GT1c and gO GT4. Fibroblasts (HFF) and epithelial cells (ARPE-19) were infected for 2 h with strains of the GT1c-group (A,B) or of the GT4-group (C,D) with 3.59 × 10⁸ encapsidated genomes/ml, which correspond to an MOI of 1 in GT1c_1. Luciferase assay was performed 48 h post infection and RLUs were determined in triplicates and three independent experiments were performed. Error bars indicate SD. Mean RLUs of the mutants were compared individually to both respective parental clones. Mutants that are significantly different to at least one of the two parental clones are marked by asterisks. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001, unpaired two-tailed t-test.

FIGURE 4

Quantitative Western blots of purified virions. Virions were subjected to reducing gel electrophoresis and analyzed by Western blot using antibodies directed against major capsid protein (MCP), and the glycoproteins gO (anti-gO antibody gO.02) and gH (anti-gH antibody AP86-SA4). The amounts of virions loaded on the gels were normalized to equal amounts of MCP. First, the parental strains GT1c and GT4 were compared with each other (A). Next, the four mutants were MCP-adjusted to each other and compared for content of gO and gH (B). Additionally, the mutants GT1c-C343S and GT4-C336S were analyzed using increased amounts of virions for better detection of gO (C). Band densities were determined relative to one reference band for each blot individually, and are shown below the blots. Mass markers are indicated on the left in kilodalton (kDa).

FIGURE 5

Replication and cytopathic effect of gO GT1c-C343S and gO GT4-C336S mutants. Fibroblasts (HFF) and epithelial cells (ARPE-19) were infected with the mutants GT1c-C34S and GT4-C336S, and their respective parental strains GT1c and GT4 at an MOI normalized to 1000–1500 RLU at 48 h post infection. Infected cells were cultured for 13 (HFF) or 15 (ARPE-19) days. At the indicated time points cell-free (A,C) and cell-associated (B,D) viral loads were assessed by qPCR. Shown are mean values of three replicates; error bars indicate SD. In (E) representative light microscopy pictures of the cytopathic effect of GT1c, GT4 and mutants are shown as seen in HFFs at day 13 and ARPE-19 at day 15 post infection. Infected HFFs of gO GT4-C336S are marked by a red, dashed line.

FIGURE 6

Comparison of gO GT1c and gO GT4 amino acid sequences. Reference sequences of genotype (GT) 1c (TB40BAC4; Protein ID: ABV71596.1) is aligned with gO GT4 sequence of HCMV strain Towne (Protein ID: KF493877.1). Putative N-glycosylation sites as predicted by NetNGlyc 1.0 are indicated by black boxes and putative O-glycosylation sites as predicted by NetOGlyc 4.0 are highlighted in gray. The black bar indicates the hydrophilic stretch as characterized previously (Zhou et al., 2013). The asterisks show the cysteine residues, GT1c-C218 or GT4-C216, and GT1c-C343 or GT4-C336. The black triangle depicts the O-glycosylation site identified by mass spectrometry (Bagdonaite et al., 2016).