Structural basis for a highly conserved RNA-mediated enteroviral genome replication.

Enteroviruses contain conserved RNA structures at the extreme 5' end of their genomes that recruit essential proteins 3CD and PCBP2 to promote genome replication. However, the high-resolution structures and mechanisms of these replication-linked RNAs (REPLRs) are limited. Here, we determined the crystal structures of the coxsackievirus B3 and rhinoviruses B14 and C15 REPLRs at 1.54, 2.2 and 2.54 Šresolution, revealing a highly conserved H-type four-way junction fold with co-axially stacked sA-sD and sB-sC helices that are stabilized by a long-range A•C•U base-triple. Such conserved features observed in the crystal structures also allowed us to predict the models of several other enteroviral REPLRs using homology modeling, which generated models almost identical to the experimentally determined structures. Moreover, our structure-guided binding studies with recombinantly purified full-length human PCBP2 showed that two previously proposed binding sites, the sB-loop and 3' spacer, reside proximally and bind a single PCBP2. Additionally, the DNA oligos complementary to the 3' spacer, the high-affinity PCBP2 binding site, abrogated its interactions with enteroviral REPLRs, suggesting the critical roles of this single-stranded region in recruiting PCBP2 for enteroviral genome replication and illuminating the promising prospects of developing therapeutics against enteroviral infections targeting this replication platform.

Structure of saguaro cactus virus 3' translational enhancer mimics 5' cap for eIF4E binding.

The genomes of several plant viruses contain RNA structures at their 3' ends called cap-independent translation enhancers (CITEs) that bind the host protein factors such as mRNA 5' cap-binding protein eIF4E for promoting cap-independent genome translation. However, the structural basis of such 5' cap-binding protein recognition by the uncapped RNA remains largely unknown. Here, we have determined the crystal structure of a 3' CITE, panicum mosaic virus-like translation enhancer (PTE) from the saguaro cactus virus (SCV), using a Fab crystallization chaperone. The PTE RNA folds into a three-way junction architecture with a pseudoknot between the purine-rich R domain and pyrimidine-rich Y domain, which organizes the overall structure to protrude out a specific guanine nucleotide, G18, from the R domain that comprises a major interaction site for the eIF4E binding. The superimposable crystal structures of the wild-type, G18A, G18C, and G18U mutants suggest that the PTE scaffold is preorganized with the flipped-out G18 ready to dock into the eIF4E 5' cap-binding pocket. The binding studies with wheat and human eIF4Es using gel electrophoresis and isothermal titration calorimetry, and molecular docking computation for the PTE-eIF4E complex demonstrated that the PTE structure essentially mimics the mRNA 5' cap for eIF4E binding. Such 5' cap mimicry by the uncapped and structured viral RNA highlights how viruses can exploit RNA structures to mimic the host protein-binding partners and bypass the canonical mechanisms for their genome translation, providing opportunities for a better understanding of virus-host interactions and non-canonical translation mechanisms found in many pathogenic RNA viruses.

Crystal structure of a highly conserved enteroviral 5' cloverleaf RNA replication element.

The extreme 5'-end of the enterovirus RNA genome contains a conserved cloverleaf-like domain that recruits 3CD and PCBP proteins required for initiating genome replication. Here, we report the crystal structure at 1.9 Å resolution of this domain from the CVB3 genome in complex with an antibody chaperone. The RNA folds into an antiparallel H-type four-way junction comprising four subdomains with co-axially stacked sA-sD and sB-sC helices. Long-range interactions between a conserved A40 in the sC-loop and Py-Py helix within the sD subdomain organize near-parallel orientations of the sA-sB and sC-sD helices. Our NMR studies confirm that these long-range interactions occur in solution and without the chaperone. The phylogenetic analyses indicate that our crystal structure represents a conserved architecture of enteroviral cloverleaf-like domains, including the A40 and Py-Py interactions. The protein binding studies further suggest that the H-shape architecture provides a ready-made platform to recruit 3CD and PCBP2 for viral replication.