To investigate the interaction between HCV core protein and NS5B RdRp, and the effect of this protein interaction on viral RNA synthesis in vitro, both full-length core and NS5B
Platelet Membrane Glycoprotein IIB Peptide (296-306) were expressed and purified from E. coli as fusion proteins with an N-terminal hexahistidine. Using highly purified full-length recombinant core and NS5B proteins, as assessed by Coomassie staining ( Fig. 1A), we performed immunoprecipitation experiments to demonstrate the direct interaction between these proteins. The core protein was immunoprecipitated with an anti-core protein antibody. As seen in Fig. 1B, immunoprecipitation of core protein co-immunoprecipitated NS5B (lane 2). We further confirmed the interaction in human hepatoma
cells by co-immunoprecipitation experiments. Huh7TR-core cells expressing core protein were transfected with pcDNA3.1-Flag-NS5B. Flag-tagged NS5B in detergent-solubilized cell lysates were immunoprecipitated with an anti-Flag antibody, and the resulting immunocomplexes were examined for the presence of core protein by immunoblot analysis using an HCV patient serum. The results shown in Fig. 1C reveal the interaction between core protein and NS5B in cellular context (lane 6).
