Abstract Human enteroviruses are responsible for diseases that range from mild conditions like the common cold to severe diseases such as poliomyelitis and myocarditis. For example, the inflammation and weakening of the heart muscle (myocarditis) caused by coxsackie B3 virus (CBV3) can lead to heart failure. Approximately 10-15 million people are infected with enteroviruses in the US each year, yet no antiviral therapies are currently available. Our long-term goal is to obtain detailed structural and biochemical information regarding the enterovirus replication process, and to use this information for the development of antiviral therapeutics and vaccines. In enteroviruses and other positive-strand RNA viruses, the RNA genome is used as a template for both protein translation and RNA synthesis, and thus these viruses need a mechanism to balance the relative extents of these two processes. Enteroviruses use a ‘cloverleaf’ four-way junction RNA structure at the 5’ ends of their genomes to serve as a binding site for the host protein, poly(rC)-binding protein 2 (PCBP2) and the virally encoded protein 3CD, and to modulate the relative levels of viral protein translation and RNA genome synthesis. The goal of the project is to understand how the cloverleaf RNA interacts with these host and virus proteins to balance protein translation and genome synthesis to ensure efficient replication. To accomplish these goals, we will determine the structure of 5’ cloverleaf of the coxsackie virus genome and characterize its interactions with PCBP2 using complementary structural, biochemical, and biophysical approaches. The structure of cloverleaf is predicted to be conserved in enterovirus, rhinovirus, and other picornaviruses, and hence our studies will inform how the 3D structure of cloverleaf RNA regulates viral replication in this large class of viruses.