Colocalization of (+) and (−) strand HCV RNAs with active translating ribosomes.

<p><b>A</b>. Huh-7.5 cells were infected with HCV at MOI = 1.5 and at 48 hours post-infection cells were left untreated or pre-treated with anisomycin (Ani) (competitive inhibitor of puromycin; 9.4 uM) followed by puromycin (Puro) labeling and digitonin extraction before fixation. Cells were processed for immunofluorescence using the anti-puromycin PMY-2A4 monoclonal antibody. Scale bar is 5 μm. <b>B</b>. Huh-7.5 cells were infected with HCV at MOI = 1.5 and at the indicated times post-infection the cells were fixed and processed for strand specific RNA detection followed by immunofluorescence staining for puromycylated ribosomes. Scale bar is 5 μm. Insets represent 10 times magnification of the merged image. Solid arrows point to (+) strand RNA colocalizing with ribosomes; arrowheads point to (−) strand RNA colocalizing with ribosomes. <b>C</b>. Quantitation of % colocalization in (B). Each error bar indicates standard deviation from 25 different images. <b>D</b>. Huh-7.5 cells were infected with HCV at MOI = 1.5 and at 6 hpi the cells were fixed and processed for strand specific RNA detection followed by immunofluorescence staining for calnexin.