Fig. 2.
GRFT interferes with direct cell-to-cell spread of HCV in vitro. (A) The effect of GRFT on direct cell-to-cell transmission of HCV was investigated by incubating HCV-infected Huh-7 cells with different concentrations (in micrograms per milliliter) of GRFTWT or GRFTMUT in agarose-containing medium. At 72 h postinfection, the cultures were immunostained with an anti-E1 antibody, and the number of HCV-positive cells per focus was enumerated in 17 foci. The experiments were performed two times. Each symbol represents the value for an individual focus, and the short horizontal lines indicate the average number of cells per infected focus. (B) Representative pictures of HCV-infected agarose overlay cultures treated with 10 μg/ml GRFTMUT (top panel) or GRFTWT (bottom panel), indicating that GRFTWT interferes with direct cell-to-cell transmission of HCV. HCV-infected cells are visualized via immunofluorescence microscopy using an E1-specific antibody. (C) Cocultures of Huh-7.5 target cells with Jc1-infected Huh-7.5-RFP-NLS-IPS were treated with an anti-HCV neutralizing antibody (nAb; 150 μg/ml) or with 10 μg/ml GRFTWT alone or in combination with the neutralizing antibodies. More than 100 foci were analyzed per condition. (D) Representative images taken from a nAb-treated coculture (top panel) showing a focus of infected target cells (green cytoplasm and blue nucleus) that surround an infected donor cell (green cytoplasm and pink nucleus). In cocultures treated with GRFTWT alone (bottom panel), the infection remains restricted to the donor cells (green cytoplasm and pink nucleus). The 10 μg/ml concentration of GRFT corresponds to 769 nM.