Exploring the structural biology of enteroviral genome replication

The Enterovirus genus comprises RNA viruses associated with various illnesses, including the common cold, poliomyelitis, acute flaccid paralysis, and myocarditis. These viruses have conserved cloverleaf-shaped RNA secondary structures at the extreme 5′ end of their genomes, which are crucial for assembling viral and host proteins, such as 3CD and PCBP2, into functional ribonucleoprotein (RNP) complexes required for genome replication. However, the high-resolution structures and mechanisms of these cloverleaf RNAs have largely remained unknown. Recently, we determined the crystal structures of five cloverleaf RNAs from different enteroviral species, revealing a highly conserved H-type four-way junction fold that positions the 3CD and PCBP2 binding sites far apart. The conserved features observed in these structures, including a long-range A•C•U base triple, enabled us to predict structural models for other enteroviruses via homology modeling, yielding models similar to the experimental structures. Our structure-guided binding analyses, using recombinantly purified full-length human PCBP2 and viral 3C proteins, along with the crystal structures of the cloverleaf-3C complexes, offered further insights into how these viruses assemble RNP complexes for their genome replication within host cells. Moreover, we demonstrated that antisense oligonucleotides could block high-affinity PCBP2-binding sites, highlighting the potential to develop therapeutics targeting this replication platform for enteroviral infections. The notable structural conservation among enteroviral cloverleafs observed in our studies underscores opportunities to develop universal therapeutics that target this viral RNP platform across multiple enterovirus infections.