Functional role of the 5′ terminal cloverleaf in Coxsackievirus RNA replication

Using cell-free reactions, we investigated the role of the 5′ cloverleaf (5′CL) and associated C-rich sequence in Coxsackievirus B3 RNA replication. We showed that the binding of poly(C) binding protein (PCBP) to the C-rich sequence was the primary determinant of RNA stability. In addition, inhibition of negative-strand synthesis was only observed when PCBP binding to both stem-loop ‘b’ and the C-rich sequence was inhibited. Taken together, these findings suggest that PCBP binding to the C-rich sequence was sufficient to support RNA stability and negative-strand synthesis. Mutational analysis of the three conserved structural elements in stem-loop ‘d’ showed that they were required for efficient negative- and positive-strand synthesis. Finally, we showed an RNA with a 5′ terminal deletion (Δ49TD RNA), which was previously isolated from persistently infected cells, replicated at low but detectable levels in these reactions. Importantly, the critical replication elements identified in this study are still present in the Δ49TD RNA.