Soluble THY-1 protein blocks HCMV entry in a dose-dependent manner.

<p>(A) HS-578T (adenocarcinoma) cells were infected with Towne-GFP in the presence of increasing amounts of soluble protein. The virus was allowed to enter for 60 min as described in the legend to Fig 2. After culture for 3 days GFP-positivity was quantified by FACS. Equal amounts of THY-1-His and gE-His proteins were used in the assays based on Micro BCA Protein analysis (Pierce, Rockford, IL) and ELISA to determine “His” units [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004999#ppat.1004999.ref046" target="_blank">46</a>]. Seven independent experiments were performed with P value < 0.0001. (B) Towne-GFP HCMV was added to MRC-5 cells at an m.o.i. of 0.1 in the presence of soluble THY-1 (0.25 ug/ml) or control protein sgE (soluble varicella-zoster virus derived gEt-His) or control filtrates obtained during purification of THY-1 protein with an Amicon Ultra centrifugal filter unit (3000 molecular weight cutoff). The virus was allowed to enter the cells for 60 min at 37°C as indicated above followed by low pH citrate buffer wash to inactivate non-internalized virus and remove the soluble proteins. Infectivity was determined by FACS analysis of GFP positive cells at day 3 post-infection. (C) HS-578T cells were infected with HSV-2-GFP (m.o.i. 0.5) in the presence of soluble THY-1 protein (0.5 μg/ml), a control protein (soluble varicella-zoster virus gE, 0.5 μg/ml) or filtrates (derived from THY-1 protein purification in which THY-1 protein was removed by an Amicon Ultra filtration column with a 3000 molecular weight cutoff). Virus infection was performed as described in (A) and then overlaid with 2% human intravenous immune globulin (IVIG) (Talecris Biotherapeutics, Research Triangle Park, NC) and GFP positive cells were determined at 24 hrs post-infection by FACS. (D) HS-578T cells were infected with Towne-GFP or Adenovirus-GFP (0.5–1.0 m.o.i.) in the presence of soluble proteins (0.5 μg/ml), filtrate control, or buffer for 60 min as described in the legend to Fig 2, and the percentage of infected cells was determined by flow cytometry 3 days after infection.