The exploitation of cellular functions and host proteins is an essential part of viral replication. The study of this interplay has provided significant insight into host cell processes in addition to advancing the understanding of the viral life-cycle. Poliovirus utilizes a multifunctional cellular protein, poly(C) binding protein 2 (PCBP2), for RNA stability, translation and RNA replication. In its cellular capacity, PCBP2 is involved in many functions, including transcriptional activation, mRNA stability and translational silencing. Using a novel protein-RNA tethering system, we establish PCBP2 as an essential co-factor in the initiation of poliovirus negative-strand synthesis. Furthermore, we identified the conserved KH domains in PCBP2 that are required for the initiation of poliovirus negative-strand synthesis, and showed that this required neither direct RNA binding or dimerization of PCBP2. This study demonstrates the novel application of a protein-RNA tethering system for the molecular characterization of cellular protein involvement in viral RNA replication.