Fig. 2: GPC of rCl13-RRRR exhibits WT-like fusion activity.
From: Site-1 protease mediated GPC processing is required for persistence of LCMV Clone 13
a GPC-RRRR had similar fusion activity compared to GPC-WT. HEK293T cells were seeded at 2.5 × 105 cells/well in a 24-well plate and 24 h later transfected with pCAGGS plasmids (1 µg) expressing LCMV GPC-WT, GPC-RRRR, GPC-RRLL, or an empty plasmid, together with 50 ng of pCAGGS-GFP. At 48 h post transfection, cell monolayers were treated with citrate buffer (pH 5.0) or PBS (pH 7.0) for 15 min, then returned to neutral pH normal growth media (DMEM containing 10% FBS) and fusion monitored over time. Once fusion activity was observed (~20 min) cells were allowed 10 min to complete fusion then fixed (4% PFA) and stained with a human monoclonal antibody to GPC (red). Fusion was visualized based on the distinctive appearance of GFP signal. b GPC-RRRR mediated fusion is inhibited by treatment with the furin inhibitor BOS-318. HEK293T cells were prepared and transfected as in (a) but also treated with BOS-318 (2 µM) or DMSO at the time of transfection, or at 46 h post transfection. Fusion assay was done at 48 h post transfection. Representative images from each condition are shown. All images were taken on a Zeiss LSM 780 Confocal laser scanning microscope.
