Poliovirus tropism in poliovirus receptor transgenic mice is limited to the central nervous system by the type I interferon system. Poliovirus is able to replication in cell cultures pre-treated for 16 hours with IFN-alpha, but not vesicular stomatitis virus, a Rhabdovirus, or encephalomyocarditis virus (EMCV), a picornavirus of the Cardiovirus genus. Although EMCV is sensitive to IFN-alpha, coinfection of cells with poliovirus and EMCV leads to EMCV replication in IFN-alpha-pretreated cells. The enteroviral 2A proteinase (2A(pro)) is essential for replication in cells pretreated with interferon, because amino acid changes in this protein render poliovirus sensitive to IFN-alpha. The addition of the poliovirus 2A(pro) gene to the EMCV genome allowed EMCV to replicate in IFN-alpha-pretreated cells. These results support an inhibitory role for 2A(pro) in the most downstream event in interferon signaling, the antiviral activities of ISGs. To identify the ISGs that inhibit poliovirus replication, an ectopic expression assay was used to screen a library of more than 350 human ISGs. Eighteen ISGs were identified that inhibit poliovirus replication, including APOL6. To understand why neural tissues are not protected by the IFN response, we evaluated the neuroblastoma cell line SK-N-SH as a model system for studying ISG induction during poliovirus replication. Our findings indicate that there is a difference in susceptibility to poliovirus infection between SK-N-SH and HeLa cells, which could be caused by variation in ISG expression. The ISG APOL6 is found at low levels in SK-N-SH cells, and overexpression markedly inhibits poliovirus replication. APOL6 may inhibit poliovirus replication by blocking the autophagy pathway, which has been suggested to be required for maturation of poliovirus particles.