Co-precipitated proteins

were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (12% gel) and subsequent immunoblot analysis. For co-immunoprecipitation, 4 μg of specific antibody and 500 μg of Huh 9-13 protein lysate were rotated for 2 hours at 4°C in a total volume of 500 μL PBS. Afterward, 2× washed protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Santa Cruz, CA) was added and incubated overnight at 4°C. Thereafter, the samples were washed 3 times with Triton-wash-buffer, resuspended and denatured in 2× SDS-PAGE buffer for 5 minutes at 95°C, supplemented at 4°C for 1 minute at 13.000 rpm and subjected to SDS-PAGE (12% gel). It has been recently reported that the benzoquinoid mTOR inhibitor ansamycin antibiotic herbimycin A, which is known to inhibit the activity of viral Src and c-Src,13-16 affects HCV replication. However, due to the propinquity of herbimycin A to geldanamycin, which binds to Hsp90 and alters its function, this finding has been linked to a potential role of Hsp90 for HCV replication, but not to a possible role of c-Src for HCV replication.3 Indeed, as shown in Fig. 1, herbimycin A concentration-dependently almost completely abrogates HCV replication when analyzed in the subgenomic replicon cell lines

Huh 9-132 (Fig. 1), Huh 5-15, and Huh 11-7 (Supporting Information Fig. 1) with maximal inhibitory effects after an incubation period of 72 hours, depending on check details the concentration used. In line with these observations, treatment with 0.1 μM herbimycin A also strongly suppressed HCV replication in Huh7.5 cells persistently infected with the JC1 strain (Fig. 1D). In contrast to HCV, replication of vaccinia virus or vesicular stomatitis virus, both of which replicate as HCV at extranuclear sites

in the cytoplasm in Huh7 cells, was resistant to treatment with 0.1 μM herbimycin A. This finding suggests that the effect of herbimycin A on HCV replication is indeed specific rather than an unselective impairment of the host cell resulting in a curtailed efficiency of viral replication many (Supporting Information Fig. 2). To assess whether the inhibition of HCV replication by herbimycin A might be due to its inhibitory effect on c-Src, three different siRNAs directed against c-Src were established and used to suppress c-Src expression and compared with GFP siRNA as a control (Fig. 2A,B). In addition, as a second approach, a commercially available c-Src siRNA pool and respective nontarget control siRNA (Dharmacon) was used (Fig. 2C,D). As shown in Fig. 2, down-regulation of c-Src expression was achieved by all siRNAs used and resulted in an effective decrease of the abundance of HCV RNA (Fig. 2A) and protein (Fig. 2B,C). Thereby suppression of viral RNA and protein expression closely coincided with the degree c-Src expression was suppressed.