Sequence analysis of NS5B for the prevalence of proline-rich motifs that represent putative sites for the interaction of NS5B with the SH3 domain of c-Src revealed two possible binding sites (Fig. 4) located between aa 347 and 355 (termed M1)

and between aa 385 and 392 (termed M2). Deletion of the C-terminal, M2 comprising part of NS5B (deletion of aa 382 to 591), but not of the N-terminal part of NS5B that contains M1 (deletion of aa 1-357) strongly reduces the interaction between NS5B and c-Src (Fig. 4), which was not the case when deletion of the C-terminal part did not include motif 2 (deletion of aa 402-591). This suggests that the M2-containing region located between aa 382-402 is important for the interaction of NS5B with this website c-Src. The fact that deletion of the N-terminal part of NS5B completely abrogates the interaction with NS5A but did not affect the interaction of NS5B with c-Src indicates that irrespective of their interaction with c-Src, NS5A and NS5B also directly interact with each other (Fig. 4). The latter observation indicates

5-Fluoracil order that, although c-Src undergoes complex formation with NS5A and/or NS5B either as one ternary complex or as two independent complexes, the interaction of NS5A and NS5B also involves direct protein–protein interactions, which is in line with previous reports.17 It has been reported that NS5A and NS5B directly interact with each other Guanylate cyclase 2C and that this complex formation of NS5A and NS5B is essentially required for efficient viral replication.10, 17 Because the data presented herein suggest that c-Src is part of this protein complex, the question was addressed whether the interaction of NS5A and NS5B is sensitive toward the tyrosine kinase inhibitor herbimycin A. As shown in Fig. 6A, treatment of Huh 9-13 cells harboring the subgenomic replicon of HCV

with herbimycin A for 14 hours results in a substantial reduction of the amount of NS5B coprecipitated with NS5A, suggesting that the interaction of NS5A and NS5B is sensitive to herbimycin A. That complex formation of NS5A and NS5B indeed requires the presence of c-Src is further substantiated by the fact that suppression of c-Src expression using specific siRNA likewise resulted in an impaired protein–protein interaction of NS5A and NS5B if analyzed by co-immunoprecipitation experiments using antibodies specifically directed against NS5A (Fig. 6B). It can be concluded from these data that c-Src is required to enhance complex formation of NS5A and NS5B, which in turn is essential for viral replication. Replication of viruses completely relies on host cell infrastructure, and therefore viruses have evolved mechanisms to control and use cellular machineries. As shown in the present study, HCV appears to exploit the cellular tyrosine kinase c-Src to achieve efficient RNA replication.