Picornaviruses have small, positive-sense RNA genomes and are traditionally described as cytoplasmic because their replication is carried out in the cytoplasm of the infected cell. However, a growing body of evidence suggests that proteins that are normally localized to the nucleus of cells have important roles in the replication of viruses in this family. In fact, picornaviruses of the enterovirus genus encode proteinases that directly target the proteins that make up the nuclear pore complex for degradation, resulting in the redistribution of nuclear proteins into the cytoplasm. To better characterize the virus-host interactions that occur throughout the course of an enterovirus infection we utilized two experimental approaches. The first and most direct approach sought to determine the proteome of poliovirus replication complexes. We developed recombinant viruses containing aptamer tag insertions within their genome, for biochemical isolation of viral RNA and associated proteins from infected cells. Despite the stable insertion of exogenous sequence specific for positive- or negative-strand purification, we were unable to isolate these RNAs. However, this work provides a template for similar studies with superior isolation techniques. Through a more indirect approach, we used quantitative protein mass spectrometry to identify proteins that both increase in abundance in the cytoplasm of human rhinovirus 16 (HRV16)-infected cells. We used molecular methods to validate the involvement of SFPQ in HRV16 replication, confirming that our cytoplasmic enrichment screen is an effective approach to reveal novel viral replication factors. In subsequent investigations, we aim to combine these distinct but complementary approaches to expand the known array of host proteins that may normally be found within the nucleus but are required for enterovirus replication. The work presented in this dissertation reinforces the idea that enteroviruses are not unambiguously cytoplasmic in nature, presents new methodologies for describing virus-host interactions in molecular detail, and provides direct evidence for a novel nuclear factor in the replication cycle of HRV16. Only through taking a more integrated view of the enterovirus-infected cell can we gain a deeper understanding of enterovirus biology and foster the development of treatments for a group of viruses that continue to burden society.