PDGF Receptor-α Does Not Promote HCMV Entry into Epithelial and Endothelial Cells but Increased Quantities Stimulate Entry by an Abnormal Pathway

Epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor-α (PDGFRα) were reported to mediate entry of HCMV, including HCMV lab strain AD169. AD169 cannot assemble gH/gL/UL128–131, a glycoprotein complex that is essential for HCMV entry into biologically important epithelial cells, endothelial cells, and monocyte-macrophages. Given this, it appeared incongruous that EGFR and PDGFRα play widespread roles in HCMV entry. Thus, we investigated whether PDGFRα and EGFR could promote entry of wild type HCMV strain TR. EGFR did not promote HCMV entry into any cell type. PDGFRα–transduction of epithelial and endothelial cells and several non-permissive cells markedly enhanced HCMV TR entry and surprisingly, promoted entry of HCMV mutants lacking gH/gL/UL128–131 into epithelial and endothelial cells. Entry of HCMV was not blocked by a panel of PDGFRα antibodies or the PDGFR ligand in fibroblasts, epithelial, or endothelial cells or by shRNA silencing of PDGFRα in epithelial cells. Moreover, HCMV glycoprotein induced cell-cell fusion was not increased when PDGFRα was expressed in cells. Together these results suggested that HCMV does not interact directly with PDGFRα. Instead, the enhanced entry produced by PDGFRα resulted from a novel entry pathway involving clathrin-independent, dynamin-dependent endocytosis of HCMV followed by low pH-independent fusion. When PDGFRα was expressed in cells, an HCMV lab strain escaped endosomes and tegument proteins reached the nucleus, but without PDGFRα virions were degraded. By contrast, wild type HCMV uses another pathway to enter epithelial cells involving macropinocytosis and low pH-dependent fusion, a pathway that lab strains (lacking gH/gL/UL128–131) cannot follow. Thus, PDGFRα does not act as a receptor for HCMV but increased PDGFRα alters cells, facilitating virus entry by an abnormal pathway. Given that PDGFRα increased infection of some cells to 90%, PDGFRα may be very useful in overcoming inefficient HCMV entry (even of lab strains) into the many difficult-to-infect cell types

Gleevec inhibits both HCMV and HSV immediate early protein expression.

<p>Normal ARPE-19 cells were treated with various concentrations of Gleevec for 1 hr at 37<b>°</b>C then incubated with: <b>A</b>) HCMV TR using 10 IU/cell or <b>B</b>) HSV-1 using 10 PFU/cell in the presence of Gleevec for 4 hr at 37<b>°</b>C. The cells were washed to remove the virus inoculum then incubated for an additional 24 hr in growth media containing the same concentration of Gleevec. HCMV-infected cells were stained for IE-86 and HSV-infected cells were stained for immediate early protein ICP4. The percent infected cells was calculated by determining the percent IE-86+ or ICP4+ cells and comparing to the number of IE-86+ or ICP4+ cells observed when a different set of cells were treated with Gleevec after the virus inoculum was removed (at 4 hr post-infection). The numbers represent the average from at least three separate fields. Error bars represent the standard deviation.</p

HCMV entry into PDGFRα expressing epithelial and endothelial does not require gH/gL/UL128–131 complexes.

<p><b>A</b>) ARPE-19 epithelial cells or <b>B</b>) HUVECs were transduced with Ad-tet-trans or Ad-PDGFRα and Ad-tet-trans for 24 hr. The cells were then incubated with either HCMV lab strain AD169 or HCMV TR mutant virus TRΔ131 at 10 IU/cell for an additional 24 hr, then fixed, permeabilized, and assayed for HCMV infection by staining cells for the HCMV IE-86 early antigen. As in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002905#ppat-1002905-g002" target="_blank">Fig. 2</a>, the percent infected cells was calculated by counting IE-86 positive cells in three separate wells involving two experiments and the average number of IE-86+ cells indicated in the lower right of each panel.</p

The PDGF ligand (PDGF-AA) and PDGFRα antibodies do not reduce HCMV infection of epithelial, endothelial cells, and fibroblasts.

<p>(<b>A</b>) ARPE-19, HUVECs, or foreskin fibroblasts (HFF) were pre-treated with PDGF-AA ligand at various concentrations at 4<b>°</b>C for 30 min then HCMV TR (10 IU/cell, 1 IU/cell for fibroblasts) was added to the cells in the presence of PDGF-AA at 4<b>°</b>C for an additional 30 min. The cells were shifted to 37<b>°</b>C for 4 hr then washed to remove the virus inoculum and fresh growth media containing the same concentration of PDGF-AA was added. After an additional 24 hr, the cells were fixed, permeabilized, and stained for HCMV IE-86. Relative entry was characterized by comparing the number of IE-86 positive cells in three separate fields with other dishes of cells not treated with PDGFR-AA. Error bars indicate the standard deviation. <b>B</b>) ARPE-19 cells, <b>C</b>) HUVE cells or <b>D</b>) human foreskin fibroblasts were incubated with various doses of PDGFRα-specific antibodies for 1 hr at 4°C then incubated with HCMV TR (10 IU/cell, 1 PFU/cell for fibroblasts) in the presence of these antibodies for an additional 1 hr at 4°C. The cells were then shifted to 37°C for 4 hr, washed to remove the virus inoculum then incubated at 37°C for 24 hr in the presence of antibodies. The cells were then stained for IE-86. Relative entry refers to the frequency of IE-86 positive cells compared to cells not treated with antibodies and was determined from at least three separate fields. Error bars indicate the standard deviation.</p

Expression of PDGFRα enhances entry of HCMV TR into epithelial and endothelial cells.

<p><b>A</b>) Human epithelial cells (ARPE-19), <b>B</b>) human endothelial cells (HUVECs), <b>C</b>) human foreskin fibroblasts (HFF), <b>D</b>) owl monkey kidney cells (OMK), and <b>E</b>) rat retinal epithelial cells (Rat-RPE) were transduced with Ad vectors expressing tet-trans, PDGFRα, or EGFR using 50 PFU/cell for each Ad vector for 24 hr. The cells were infected with HCMV TR using 10 IU/cell and 1 IU/cell for HFF cells then fixed, permeabilized, and stained for HCMV IE-86 immediate early protein. The percent infected cells was calculated by counting the number of IE-86+ cells in three fields from three separate wells involving two separate experiments, comparing to the total number of cells in each field then averaging the numbers. Standard deviations are indicated.</p

Reduction in expression of PDGFRα in ARPE-19 epithelial cells does not reduce HCMV infection.

<p><b>A</b>) ARPE-19 cells, ARPE-19 cells transduced with a retrovirus expressing an shRNA targeting PDGFRα (ARPE-19 KD), or ARPE-19 cells transduced with a retrovirus causing overexpression of PDGFRα (ARPE-19α) were analyzed by staining western blots with a PDGFRα–specific polyclonal antibody. As a loading control, the same membrane was also probed for the cellular protein, β-actin with the mouse MAb AC-15. Note that the same gel was used to analyze PDGFRα in all three cell types, but a much stronger PDGFRα signal was expected for ARPE-19α cells and, thus, we separated the lanes in the figure. <b>B</b>) ARPE-19, ARPE-19 KD, and ARPE-19α cells were incubated with HCMV TR using 10 IU/cell for 24 hr then the cells were fixed, permeabilized, and stained for HCMV IE-86. The percent cells infected was derived by counting IE-86+ cells in at least three separate fields. Error bars represent the standard deviation.</p

Characterization of Ad vectors expressing human PDGFRα and EGFR.

<p>(<b>A</b>) ARPE-19 epithelial cells were transduced with Ad-tet-trans alone (100 PFU/cell) or Ad-PDGFRα and Ad-tet-trans (50 PFU/cell of each) or Ad-EGFR and Ad-tet-trans (50 PFU/cell of each) for 24 hr then radiolabeled with [³⁵S]-methionine-cysteine for 4 hr. The cells were lysed and the PDGFRα or EGFR proteins immunoprecipitated with a goat polyclonal antibody AF-307-NA or mouse MAb LA1, respectively. The precipitated proteins were analyzed by SDS-PAGE. Molecular weight markers are indicated on the left. IP refers to the protein immunoprecipitated. (<b>B</b>) ARPE-19 epithelial, human foreskin fibroblasts (HFF), and human endothelial cells (HUVECs) were transduced with Ad vectors expressing tet-trans, PDGFRα, or EGFR as described above, incubated for 24 hr then fixed and stained with the PDGFRα-specific antibody AF-307-NA or EGFR-specific antibody LA-1 followed by a FITC-conjugated donkey anti-goat (Molecular Probes) or a Dylight 594-congugated goat anti-mouse secondary antibody.</p

The pathway of HCMV entry into PDGFRα expressing epithelial cells is different from that involving normal ARPE-19 cells.

<p><b>A</b>) ARPE-19 cells were transduced with Ad-tet-trans alone (Normal) or Ad-PDGFRα and Ad-tet-trans (PDGFRα expressing) for 24 hr at 37<b>°</b>C. The cells were incubated with 0.2% DMSO (No drug), 50 mM ammonium chloride (NH₄Cl), 150 µM dynasore (Dyna), 30 µM chlorpromazine (Cpz), 30 µM Rottlerin, or 75 µM EIPA for 1 hr at 37<b>°</b>C then HCMV TR (10 IU/cell) was added for an additional 4 hr in the presence of drugs. The cells were then washed to remove the virus inoculum then incubated in media containing 10% FBS and the same concentration of drug for 24 hr at 37<b>°</b>C. The relative level of HCMV infection was measured by comparing the number of IE-86 positive cells in drug-treated cells with cells treated with 0.2% DMSO. The bars represent the averages from three separate wells from one experiment with standard deviations shown. Other experiments produced very similar data. <b>B</b>) ARPE-19 or ARPE-19α cells were treated with either 0.2% DMSO or with 30 µM chlorpromazine (Cpz) for 1 h at 37<b>°</b>C then incubated with media containing Alexa-fluor-488 transferrin and DMSO or chlorpromazine for 30 min at 37<b>°</b>C. The cells were placed on ice, washed briefly with citrate buffer, pH 3.0 to remove cell surface transferrin, fixed, the nuclei stained with DAPI, then analyzed by fluorescent microscopy. <b>C</b>) ARPE-19 or ARPE-19α cells seeded on glass coverslips were incubated with UL32-EGFP-HCMV using 10 IU/cell for 1 hr at 37°C then the cells washed and incubated for an additional 2, 4, 12, 16 hr in growth media before the cells were fixed, nuclei counterstained with DAPI, and cells analyzed by fluorescence microscopy. Cells incubated for 24 hr were fixed, permeabilized, and stained for the HCMV IE-86 early antigen along with an Alexa-fluor-594 secondary antibody and then counterstained with DAPI. Coverslips were mounted on glass slides and a 0.2 µm section of the Z-stack (including the central plane of the nucleus) was captured.</p