Structural Basis For Poliovirus Cloverleaf-Mediated Regulation Of Genome Replication

Poliovirus is a member of the Enterovirus genus, members of which are characterized by small, non-enveloped capsids containing short, positive-sense single-stranded RNA genomes. During the viral replication cycle, it is critical that the virus generates the appropriate amounts of viral proteins and genomic RNA. Upon entry in the cell, the viral genome is first used as a template for translation of viral genes and then as a template for genome replication. Regulation of these two processes is mediated by a series of six stem-loops in the 3’ untranslated region of the genomic RNA. Stem-loops II-VI comprise an internal ribosomal entry sequence (IRES), which makes cap-independent translation possible. Stem-loop I, also known as the cloverleaf, serves as a switch to shut off translation, and promote negative strand RNA synthesis. The cloverleaf has previously been shown to form a three-component complex with the viral protein product 3CD (a fusion of 3C protease and 3D RNA polymerase) and host-protein Poly-C binding Protein 2. The formation of this complex is critical for the initiation of negative-strand RNA synthesis. In order to investigate the function and role of the cloverleaf, we report the X-ray crystal structure of the poliovirus cloverleaf RNA. Additionally, we report binding studies and conceptual modeling to characterize the interaction between the cloverleaf RNA, 3CD and Poly-C Binding Protein 2.