1E). From these results, we assumed that the differences in ADK expression were involved in the dramatic differences in RBV sensitivity between the two cell lines. To address this assumption, we focused on the ADK-short in the following study; hereafter, ADK-short is designated as ADK. To evaluate the hypothesis that ADK controls the anti-HCV activity of RBV, we first examined the effect of ABT-702, an ADK inhibitor, on the anti-HCV activity of RBV. The results revealed that ABT-702 cancelled Navitoclax the activity of RBV in ORL8 cells in a dose-dependent manner (Fig. 2A). Furthermore, we demonstrated that the activity of RBV was

cancelled in ADK-knockdown ORL8 cells (Fig. 2B). These results suggest that the inhibition of ADK in ORL8 cells converts them from an RBV-sensitive phenotype to an RBV-resistant phenotype. To directly demonstrate the involvement of ADK, we first prepared OR6 cells

stably expressing ADK (OR6-ADK) (Fig. 2C). We were able to demonstrate that the OR6-ADK cells were dramatically converted from an RBV-resistant phenotype with an EC50 value of more than 100 µM to an RBV-sensitive phenotype with an EC50 value of 2.6 µM (Fig. 2D). We next examined whether or not the GTP reduction or IMP accumulation observed in ORL8 cells treated with RBV (Fig. 1A,B) occurs in OR6-ADK cells. The results revealed that the GTP reduction and IMP accumulation in RBV-treated OR6-ADK cells were more pronounced than in RBV-treated ORL8 cells (Supporting Fig. 3A,B). Because OR6 is a clonal cell line harboring genome-length Hydroxychloroquine HCV RNA, we used a polyclonal cell line (sOR) harboring HCV replicon RNA[9] to prepare sOR-ADK cells stably expressing ADK (Supporting Fig. 3C) and examined their sensitivity to RBV. sOR-ADK cells were also dramatically converted from an RBV-resistant phenotype with an EC50 value of more than 100 µM to an RBV-sensitive find more phenotype with an EC50 value of 6.0 µM (Supporting Fig. 3D). In addition, ORL8-ADK cells stably overexpressing ADK also showed EC50 values ranging from 13.2 to 1.2 µM (Supporting Fig. 3E). Furthermore, we demonstrated that the anti-HCV activity detected in OR6-ADK

cells was also cancelled by ABT-702 treatment in a dose-dependent manner (Fig. 2E). Considering these results together, we conclude that ADK is a key determinant for the anti-HCV activity of RBV. To clarify the mechanism underlying the difference in ADK expression between OR6 and ORL8 cells, we first examined the nt sequences of up to several kb upstream from the transcription start point estimated from NM_001123 (31-OCT-2010) using the data of AL731576. Several possible transcription elements, such as the GC box (−12 and −187 of ADK gene), p53 response element (−252 and −585), and heat shock element (−559, −971, −1486, and −1797) were detected in up to approximately 2 kb upstream from the estimated transcription start point, but not in more 2 kb.