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

By Adam L. Vanarsdall (307887), Todd W. Wisner (307889), Hetian Lei (307891), Andrius Kazlauskas (307893) and David C. Johnson (307895)

Abstract

<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 (NH<sub>4</sub>Cl), 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

Topics: Biological Sciences, microbiology, Molecular cell biology, pathway, hcmv, expressing, epithelial, cells, involving, arpe-19
Year: 2013
DOI identifier: 10.1371/journal.ppat.1002905.g009
OAI identifier: oai:figshare.com:article/248991
Provided by: FigShare
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