Members of the Enterovirus genus of picornaviruses are responsible for a wide range of diseases, including myocarditis, paralytic poliomyelitis, the common cold, and encephalitis. In the cytoplasm of infected cells, these viruses (e.g., coxsackievirus, poliovirus, EV71, human rhinovirus) replicate their positive-sense RNA genome via a two-step process. First, the positive-sense genomic RNA is used as a template to synthesize a complementary negative RNA strand. This intermediate negative RNA strand then serves as a template to make multiple positive RNA strands that function both as mRNA and as genomic RNAs for new virus particles. To date, most studies of enterovirus RNA replication have focused on the first step: synthesis of the negative-strand RNA. Experiments proposed in this application will focus on the second, and arguably more central, step: synthesis of the multiple positive RNA strands needed to form new virus particles. Specifically, these studies will focus on a predicted RNA cloverleaf-like structure that forms at the 3’ end of the negative strand, the so-called 3’-cloverleaf (3’-CL). The 3’-CL is thought to form a platform that serves as a crucial player and binding site for different virus and host proteins that together initiate the 2nd RNA replication step. The first aim of the proposal will implement state-of- the-art approaches such as ChIRP-MS and RNA affinity methods coupled with mass spectrometry to identify host and virus proteins that interact with the 3’-CL of coxsackievirus B3 (CVB3). For the second aim, the three- dimensional structure of the predicted 3’-CL of CVB3 RNA will be solved, providing the first high resolution image of this critical replication platform. This will be done via a state-of-the-art combined NMR/Small Angle X-ray Scattering (SAXS) approach. In the final aim, CLIP-seq analysis and NMR-based chemical shift perturbation will be used to map the binding of the identified proteins onto the 3’-CL RNA structure. These studies will significantly advance our understanding of the key 2nd step of enterovirus RNA replication and identify novel therapeutic targets that may provide multiple opportunities to combat enterovirus infections by blocking the assembly of viral RNA replication complexes.