In a further analysis of drug mechanism, our study demonstrates that the antiviral activity of idarubicin and other closely related anthracyclines is derived from enhancing the activity of the type I IFN signaling pathway. Our data further show that the enhancing effect of idarubicin is based on ISRE activation and ISG expression independent of IFN production or IFN-IFN-receptor interaction, since the effect of idarubicin is unchanged by IFNreceptor blockade. These findings suggest that idarubicin activation of the ISRE is due to an action in the IFN signaling pathway distal to ligand-receptor binding, e.g., at the level of receptorassociated JAK kinases or further downstream at the formation, transport, binding, and/or assembly of the ISRE transcriptional complex. In that regard, anthracyclines are known to inhibit DNA and RNA synthesis by intercalating between base pairs of the DNA/RNA strand to prevent replication, but whether this mechanism can affect ISRE or other gene promoter elements still needs to be defined. The present screening approach overcomes the uncertainty in molecular mechanism by using a phenotypic (rather than a target-based) screening approach and thereby captures compounds that increase the activity of the IFN signaling pathway by either established or undefined mechanisms.