The pentameric complex drives immunologically covert cell-cell transmission of wild-type human cytomegalovirus

Abstract

Human cytomegalovirus (HCMV) strains that have been passaged in vitro rapidly acquire mutations that impact viral growth. These laboratory-adapted strains of HCMV generally exhibit restricted tropism, produce high levels of cell-free virus, and develop susceptibility to natural killer cells. To permit experimentation with a virus that retained a clinically relevant phenotype, we reconstructed a wild-type (WT) HCMV genome using bacterial artificial chromosome technology. Like clinical virus, this genome proved to be unstable in cell culture; however, propagation of intact virus was achieved by placing the RL13 and UL128 genes under conditional expression. In this study, we show that WT-HCMV produces extremely low titers of cell-free virus but can efficiently infect fibroblasts, epithelial, monocyte-derived dendritic, and Langerhans cells via direct cell-cell transmission. This process of cell-cell transfer required the UL128 locus, but not the RL13 gene, and was significantly less vulnerable to the disruptive effects of IFN, cellular restriction factors, and neutralizing antibodies compared with cell-free entry. Resistance to neutralizing antibodies was dependent on high-level expression of the pentameric gH/gL/gpUL128-131A complex, a feature of WT but not passaged strains of HCMV.

Keywords: HCMV; cell–cell spread; herpesvirus; immune evasion; virology.

Fig. 1.

Characterization of cell–cell infection. (A) Diagrammatic representation of the method used to grow WT Merlin in vitro without risk of mutation. (B) HFFFs were infected with variants of the Merlin BAC containing mutations in UL128 and RL13, WT (tet-regulated) UL128, or WT (tet-regulated) UL128 and WT (tet-regulated) RL13 [multiplicity of infection (MOI) = 5]. Virus was titrated from supernatants or sonicated cell preparations. Cell-released virus and cell-associated virus represent the total infectivity obtained from cells or supernatant, respectively, over the course of the experiment. (C) HFFFs infected with Merlin-expressing IE2-GFP (MOI = 5; 72 h postinfection) were stained with DDAO and then cocultured with either DCs or RPE-1 cells at the indicated ratios. Samples were analyzed by flow cytometry after a further 72 h to determine the percentage of infected DCs or RPE-1 cells (DDAO^–/GFP⁺). Representative data are shown. (D) DCs were cocultured with Merlin-infected HFFFs (MOI = 5; 72 h postinfection) for the indicated time periods. Nonadherent cells were then removed and incubated alone before flow cytometric analysis 72 h after the start of coculture. In some experiments, a transwell was placed between the DCs and HFFFs, and the cultures were incubated for 72 h. Alternatively, DCs were infected via the cell-free route using virus harvested from Merlin-infected HFFF supernatants and incubated in isolation for 72 h. Virus lacking UL128–131A expression (UL128^–) was used in one well. Representative data are shown.

Fig. 2.

Cell–cell spread is resistant to neutralizing antibodies. (A and B) Cell-free Merlin-GFP was incubated for 1 h with specific antibodies (light blue) against gB (C23; 3.6 µg/mL) or UL130 and UL131A (a 1:50 dilution of a 50:50 mixture of polyclonal rabbit sera raised against peptides from UL130 and UL131A), Cytotect (purple), or serum from donors testing seronegative (white) or seropositive (black) for HCMV (1:50). HFFFs (A) or ARPE-19 cells (B) were then infected for 2 h, overlaid, and incubated for 2 wk. Infected cells were quantified by plaque assay. Percentage of neutralization was calculated relative to the negative control (no antibody). (C–E) HFFFs were infected with Merlin-GFP (MOI = 5), then stained with DDAO after 72 h, and incubated with HFFFs (C), ARPE-19 cells (D), or DCs (E) in the presence of antibodies or sera as described in A and B. Infected cells were quantified by flow cytometry 48 h after the start of coculture. Percentage of neutralization was calculated relative to the negative control (no antibody). (F) HFFFs were infected with Merlin-GFP (MOI = 5), then stained with DDAO after 72 h, and incubated with HFFFs, ARPE-19 cells, or DCs in the presence of the indicated concentrations of Cytotect. Infected cells were quantified by flow cytometry 48 h after the start of coculture. (G) Cell-free Merlin was incubated with the indicated concentrations of Cytotect for 30 min. HFFFs, ARPE-19 cells, or DCs were then infected for 2 h. After a further 24 h, cells were fixed and stained for IE-1. Infected cells were quantified by flow cytometry. Percentage of neutralization was calculated relative to the negative control (no Cytotect). (H) The concentration of Cytotect that inhibited infection by 50% was calculated from F and G.

Fig. 3.

Levels of the pentameric complex correlate with resistance to neutralizing antibodies. (A) ARPE-19 cells were infected at 60 pfu/well with either TB40-BAC4 or Merlin and incubated for 21 d in the presence of seronegative or seropositive sera (1:50). Cells were imaged after fixing and staining for IE-1. (B and C) ARPE-19 cells were infected with either TB40-BAC4 (B) or Merlin (C) and incubated for 21 d in the presence of Cytotect (purple) or seronegative (white) or seropositive (black) sera (1:50). Plaque size was measured after fixing and staining for IE-1. (D) ARPE-19 cells were infected with 60 pfu/well of Merlin, Merlin-UL128^G>T, or TB40-BAC4 and incubated for 21 d in the presence of the indicated concentrations of Cytotect. Plaque size was measured after fixing and staining for IE-1. Percentage of inhibition was calculated relative to the negative control (no Cytotect). (E) Cell-free Merlin, Merlin-UL128^G>T, or TB40-BAC4 were incubated with the indicated concentrations of Cytotect for 30 min. ARPE-19 cells were then infected for 2 h and incubated for a further 24 h. Infected cells were counted after fixing and staining for IE-1. Percentage of neutralization was calculated relative to the negative control (no Cytotect). (F) The concentration of Cytotect that inhibited plaque formation or cell-free infection by 50% was calculated from D and E. (G) Cell-free Merlin, Merlin-UL128^G>T, or TB40-BAC4 were incubated with the indicated concentrations of Cytotect for 30 min. HFFFs were then infected for 2 h and incubated for a further 24 h. Infected cells were counted after fixing and staining for IE-1. Percentage of neutralization was calculated relative to the negative control (no Cytotect). Error bars represent SEM.

Fig. 4.

Cell–cell spread in LCs is highly efficient and resistant to IFNα. (A) Immature DCs or LCs were incubated for 24 h with cell-free preparations of TB40-BAC4 or Merlin. The percentage of infected cells was calculated by microscopy after staining with a mouse anti–IE-1 antibody and DAPI. (B) DCs or LCs were incubated for 4 h or 48 h at a 1:1 ratio with Merlin-GFP–infected HFFFs (MOI = 5; 72 h postinfection) or cell-free Merlin-GFP. Nonadherent cells were then removed and incubated alone before flow cytometric analysis 72 h after the start of coculture or cell-free infection. (C) DCs or LCs were cultured for 24 h in the presence or absence of IFNα and then incubated for 4 h or 48 h with Merlin-GFP–infected HFFFs (MOI = 5; 72 h postinfection) or cell-free Merlin-GFP. Nonadherent cells were then removed and incubated alone before flow cytometric analysis 72 h after the start of coculture or cell-free infection. Assays were performed in quadruplicate. **P < 0.01, ****P < 0.0001 (ANOVA). Error bars represent SEM.