Fig 6.

Colocalization and coimmunoprecipitation of stress granule proteins with HCV viral proteins during infection. (A) Huh-7 cells were infected with JFH-1 D183 virus at a high multiplicity of infection (MOI = 5). Seventy-two hours later, the cells were fixed and processed by immunofluorescence for the detection of G3BP1, TIA-1, or TIAR (in orange) and viral NS5A (in red) proteins. Lipid droplets (in green) were stained using LipidTOX from Invitrogen. Images displayed are representative pictures of single 0.35-μm z-sections taken in a confocal microscope at a magnification of ×63. Nuclei are displayed in blue. The staining of each channel and the merge and the colocalization mask channel between stress granule and NS5A proteins are shown. These results were confirmed in at least 2 independent experiments. (B) Huh-7 cells were infected as described for panel A and were subjected to immunoprecipitations using G3BP1-, TIA-1-, and TIAR-specific antibodies and isotype controls 72 h later, as indicated in Materials and Methods. Detection of target proteins as well as viral core, NS4A, NS4B, NS5A, and NS5B proteins was carried out in the immunoprecipitated material (IP) and the unbound material in the supernatant (S). Gels were loaded using 5% of the starting material (Input), 20% of the immunoprecipitated material (IP), and 5% of the unbound supernatant (S). These results were confirmed in 2 independent experiments.