Structures and interactions of the enteroviral replication platform

Abstract

The Enterovirus genus includes RNA viruses responsible for several human diseases, such as the common cold, poliomyelitis, acute flaccid paralysis, and myocarditis. These viruses contain conserved RNA structures at the extreme 5′ end of their genomes that recruit essential viral and host proteins, such as 3CD and PCBP2, to promote viral genome replication. This dissertation investigated the high-resolution structures of REPLRs from different humaninfecting enteroviral species and uncovered a conserved structural basis of enteroviral genome replication, illuminating promising opportunities to develop universal anti-enteroviral therapeutics targeting this platform. First, two crystal structures of the CVB3 REPLR were determined. In these crystals, the REPLR RNA folded into an antiparallel H-type four-way junction comprising four subdomains, sA, sB, sC and sD, with co-axially stacked sA-sD and sBsC helices and stabilized by a tertiary interaction between a conserved adenine within the sCloop and the sD-helix. Using NMR, mutagenesis, phylogenetic, and 3C and PCBP2 binding studies, this research also revealed that these crystal structures represent a conserved architecture of enteroviral REPLRs, including the sC-sD interactions and the H-shaped architecture provides a ready-made platform to recruit 3CD and PCBP2 for viral replication. Second, the crystal structures of the rhinoviruses B14 (RVB14) and rhinoviruses C15 (RVC15) REPLRs were determined. These structures revealed a highly conserved H-type fourway junction almost identical to the CVB3 REPLR, including the sC-sD long-range interactions. Such conserved features observed in the crystal structures also allowed us to predict the models of several other five enteroviral REPLRs using homology modeling. The structure-guided binding studies with full-length human PCBP2 showed that two previously proposed binding sites within REPLRs reside proximally and bind a single PCBP2 molecule. The DNA oligos complementary to the REPLRs abrogated their interactions with the PCBP2, illuminating promising prospects for developing therapeutics against enteroviral infections targeting this replication platform. Third, the structural basis of the enteroviral REPLR-3CD interactions was investigated using X-ray crystallography and other biophysical methods. 3CD is an enteroviral protein (fusion of 3C protease and 3D RNA-dependent RNA polymerase) required for viral RNA synthesis during replication. The crystal structures of both intact REPLR-3C and isolated sD-3C complexes were determined using CVB3 REPLR as a model system. These structures revealed that the sD stem-loop is the sole determinant for binding two 3C monomers, with each monomer recognizing the lateral surface of the sD helix. Binding studies with structure-guided REPLR and 3C mutants further clarified the roles of specific nucleotides and residues involved in the interactions between REPLR and 3C, explaining earlier virological observations. Comparative structural and binding studies of 3C, 3D, and 3CD with REPLRs from seven different enteroviral species further showed that while the 3D domain does not contribute to REPLR binding, the sD sequence and its structural pattern govern 3CD-REPLR interactions through the 3C domain. Although several more aspects of this RNA-based enteroviral genome replication need to be investigated, this research has set an initial foundation for pursuing structure-guided studies of enteroviral replication in vivo.