Equine herpesvirus type 1 (EHV-1), a member of the reporter cassette in place of the gI and gE genes (15). to the cells at a multiplicity of contamination (MOI) of 100. Computer virus was allowed to attach to the cells for 1 h at 4C. Computer virus was removed from the cells, and DMEM, prewarmed to 37C, was added. At 0 and 15 min post-temperature shift, medium was removed and the cells Mouse monoclonal to CD62P.4AW12 reacts with P-selectin, a platelet activation dependent granule-external membrane protein (PADGEM). CD62P is expressed on platelets, megakaryocytes and endothelial cell surface and is upgraded on activated platelets.This molecule mediates rolling of platelets on endothelial cells and rolling of leukocytes on the surface of activated endothelial cells were fixed with 2.5% glutaraldehyde (Sigma, St. Louis, MO). The specimens were rinsed in 0.1 M phosphate-buffered saline and then postfixed in 1% OsO4 with 0.1% potassium ferricyanide. Samples were dehydrated stepwise for 15 min each with 30%, 50%, 70%, and 90% ethanol and then embedded in Epon (dodecenyl succinic anhydride, nadic methyl anhydride, scipoxy 812 resin, and dimethylaminomethyl; Energy Beam Sciences, East Granby, CT). Semithin sections were cut on a Reichart Ultracut microtome, stained with 0.5% toluidine blue (Fisher Scientific, Pittsburgh, PA), and examined under a light microscope. Ultrathin sections were stained with 2% uranyl acetate and Reynold’s lead citrate and examined on a JEOL 1011 transmission electron microscope. At least 15 individual images of internalized virion particles were captured for each cell type. Images were captured using transmission at a magnification of 60,000. Infectious recovery assay. Cells (4 105) in a 24-well plate were washed with ice-cold medium and placed on ice for 5 min. L11gIgE, at an MOI of 10, was incubated on the cells at 4C for 2 h. Cells were washed once with cold DMEM and then incubated with DMEM which was prewarmed to 37C. At each time point, cells were washed with glycine (pH 3.0) for BRL 52537 HCl 30 s, washed once with DMEM, and harvested. Computer virus samples were freeze-thawed once and then sonicated three occasions for 15 s each. Computer virus harvested at each time point was titrated on RK13 cells. Triplicate samples were assessed for each time point. Inhibition assays. Cells (4 104) were mock treated or treated with increasing amounts of inhibitory drugs for 30 min at 37C and then infected with EHV-1 (L11gIgE), HSV-1 (QOZHG), or VSV-GFP for 6 h in the presence of the drugs. At 6 h p.i., cells were fixed with 0.5% glutaraldehyde and stained with X-Gal (5-bromo-4-chloro-3-indolyl–d-galactopyranoside; Research Products BRL 52537 HCl Intl. Corp., Mt. Prospect, IL), or ONPG (reporter gene for 6 h in the continuous presence of the drug (Fig. ?(Fig.3A).3A). As controls, ED and RK13 cells were similarly treated with BFLA and infected with EHV-1. VSV contamination of CHO-K1 cells in the presence or absence of the drug was included as a positive control of BFLA activity. The results showed a reduction in the number of CHO-K1 cells infected with EHV-1 in the presence of BFLA compared to that for cells that were not treated with the drug. No difference was observed in the number of infected ED or RK13 cells in the presence or absence of BFLA, while VSV contamination was completely inhibited in the presence of BFLA. FIG. 3. Effect of BFLA on EHV-1 entry. (A) CHO-K1, ED, or RK13 cells were mock treated (left panels) or treated with 200 nM of BFLA (right panels) for 30 min at 37C and then infected with EHV-1 (L11gIgE) or VSV-GFP (CHO-K1 cells; bottom … To quantify the reduction of EHV-1 contamination on CHO-K1 cells after BFLA treatment, an ONPG assay was employed. CHO-K1 cells plated in triplicate were treated with BFLA and infected with EHV-1 as described above, and -galactosidase manifestation was quantitated 6 h later (Fig. ?(Fig.3B).3B). The results showed that -galactosidase manifestation decreased with increasing concentrations of BFLA added to the cells. At the highest concentration tested, EHV-1 contamination of CHO-K1 cells was inhibited by 55%. While complete inhibition was not observed in this assay, these data suggest that efficient EHV-1 contamination of CHO-K1 cells requires a decrease in pH. EHV-1 entry into CHO-K1 cells BRL 52537 HCl does not require clathrin or caveolae. Many viruses enter cells through either clathrin-mediated (31, 33, 34) or caveola-dependent (46) endocytosis. To investigate which, if either, of these pathways is usually utilized by EHV-1 for contamination of CHO-K1 cells, specific inhibitors of these pathways were used. Chlorpromazine, which prevents the assembly of BRL 52537 HCl clathrin-coated pits (65), has been used extensively to prevent clathrin-mediated uptake of viruses (26, 27, 35, 49), and nystatin, a cholesterol-sequestering drug, is usually commonly employed to block.