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. 2008 Sep 16;105(37):14118-23.
doi: 10.1073/pnas.0804365105. Epub 2008 Sep 3.

HCMV gH/gL/UL128-131 interferes with virus entry into epithelial cells: evidence for cell type-specific receptors

Brent J Ryckman  1 , Marie C Chase, David C Johnson
Affiliations

HCMV gH/gL/UL128-131 interferes with virus entry into epithelial cells: evidence for cell type-specific receptors

Brent J Ryckman et al. Proc Natl Acad Sci U S A. .

Abstract

Human cytomegalovirus (HCMV) forms two different membrane protein complexes, gH/gL/gO and gH/gL/UL128/UL130/UL131, that function in different cell types. gH/gL/gO appears to be important for HCMV entry into or spread between fibroblasts, processes that occur at neutral pH. We demonstrated that HCMV entry into epithelial and endothelial cells requires gH/gL/UL128-131 and involves endocytosis and low pH. A complex of all five HCMV proteins, gH, gL, UL128, UL130, and UL131, is the functionally important mediator of this entry pathway into epithelial/endothelial cells. Here, we report that expression of gH/gL/UL128-131 in ARPE-19 epithelial cells causes the cells to be resistant to HCMV infection. Another HCMV glycoprotein, gB, did not interfere, and expression of all five gH/gL/UL128-131 proteins was required for this interference. gH/gL/UL128-131 interference was at the stage of virus entry into cells rather than the initial adsorption onto cell surfaces or after-entry defects. By contrast, expression of gH/gL/UL128-131 in primary human fibroblasts did not block HCMV infection. Previously, interference by retrovirus and herpes-simplex-virus entry mediators resulted from sequestration or obstruction of receptors. We concluded that epithelial cells express gH/gL/UL128-131 receptors that mediate HCMV entry. Fibroblasts either lack the gH/gL/UL128-131 receptors, the receptors are more numerous, or fibroblasts express other functional receptors.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Inhibition of HCMV infection of epithelial cells, but not human fibroblasts, by expression of gH/gL/UL128–131. ARPE-19 cells (A–F) or primary human fibroblasts (G–L) were left untreated (A, D, G, and J) or were transduced for 12 h with 50 PFU per cell of AdGFP (B, E, H, and K) or 10 PFU per cell each of AdgH, AdgL, AdUL128, AdUL130, and AdUL131 (C, F, I, and L). Cells were then infected with HCMV TR (3 PFU per cell) and stained for HCMV IE86 48 h later (A–C and G–I) or inoculated with 1 PFU per cell of HSV-1 for 12 h and then stained with HSV capsid protein VP5-specific antibodies (D–F and J–L).
Fig. 2.
Fig. 2.
Comparison of the interference mediated by gH/gL/L128–131, gH/gL, gB, and other HCMV proteins in epithelial cells and fibroblasts. APRE-19 (A) or primary human fibroblasts (nHDF) (B and C) cells were transduced for 12 h with the indicated combinations of Ad vectors. In A and B, each Ad vector was delivered at 10 PFU per cell, whereas in C, each Ad vector was used at 90 PFU per cell. The total dose of Ad vectors in each dish of cells was made the same by the addition of AdGFP. The cells were infected with HCMV TR (3 PFU per cell) and stained for HCMV IE86 protein 48 h later, costaining nuclei with DAPI. For each condition, at least 350 cells were counted in each of three random fields, and the average number of IE86 positive cells was normalized to the average number of infected cells observed in monolayers expressing GFP only. Error bars indicate the standard deviation from the mean. Results shown are representative of three separate experiments.
Fig. 3.
Fig. 3.
Ad vector expression of gB and gH/gL/UL128–131 in epithelial cells and fibroblasts. ARPE-19 epithelial cells and primary human fibroblasts (nHDF) were transduced for 48 h with 10 or 90 PFU per cell of AdgB (A) or AdgH + AdgL + AdUL128 + AdUL130 + AdUL131 (B–F). Cells were labeled with [35S]-methionine/cysteine, and HCMV glycoproteins were immunoprecipitated and analyzed by SDS/PAGE. Note that gL and UL130 coprecipitate (C and E, arrowheads). Numbers below each gel indicate the relative band intensities based on phosphorimager analysis.
Fig. 4.
Fig. 4.
Adsorption of HCMV particles onto epithelial cells expressing gH/gL/UL128–131. ARPE-19 cells were transduced for 12 h with 50 PFU per cell of AdGFP or 10 PFU per cell of each of AdgH, AdgL, AdUL128, AdUL130, and AdUL131. Cells were incubated with purified 3H–thymidine-labeled HCMV TR particles (representing ≈0.4 PFU per cell) at 4°C for 2 h and washed extensively, and then radioactivity bound to cells was quantified. Shown is the average number of cell-associated radioactive counts (as a percentage of input) in three experiments. Error bars represent the standard deviation from the mean.
Fig. 5.
Fig. 5.
Expression of gH/gL/UL128–131 blocks entry into epithelial cells. ARPE-19 cells were transduced for 12 h with 50 PFU per cell of AdGFP or 10 PFU per cell each of AdgH, AdgL, AdUL128, AdUL130, and AdUL131 and then incubated on ice with HCMV TR (3 PFU per cell) for 2 h. The cells were then warmed to 37°C, treated with either 44% PEG or PBS for 30 sec, washed extensively, and incubated for an additional 48 h at 37°C. The cells were fixed and stained for HCMV IE86 protein, and nuclei were stained with DAPI. Infected cells were quantified as described in Fig. 2. Results shown are representative of three separate experiments.
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