Insights into subgenomic RNA synthesis in coronaviruses from structural and biophysical studies

Abstract

The 5’ untranslated region (UTR) of coronaviral genomes contains cis-acting sequences necessary for replication, transcription and translation. A consensus secondary structural model of the 5' 140 nucleotides of the 5' UTRs of nine coronaviruses (CoVs) derived from all three major CoV groups is presented and characterized by three major stem loops, SL1, SL2 and SL4. SL2 is conserved in all CoVs, typically containing a pentaloop (C47-U48-U49-G50-U51 in MHV) stacked on a 5-bp stem, with some sequences containing an additional U 3' to U51. NMR structural studies of SL2 hairpin reveal that SL2 adopts a U-turn-like conformation. Parallel molecular genetic experiments reveal that SL2 plays an essential role in sgRNA synthesis as does SL1. We observe strong genetic selection against viruses that contain a deletion of A35, an extrahelical nucleotide that destabilizes SL1, in favor of genomes that contain a diverse panel of destabilizing second-site mutations, due to introduction of a collection of non-canonical base pairs near the deleted A35. Viruses containing destabilizing SL1-∆A35 mutations also contain one of two specific single nucleotide mutations in the 3' UTR. Thermal denaturation and imino proton solvent exchange experiments reveal that the lower half of SL1 is unstable and that second-site SL1-∆A35 substitutions recover one or more features of the wild-type SL1. We propose a "dynamic SL1" model that supports viral replication; these characteristics of SL1 appear to be conserved in other coronaviral genomes. The coronaviral nucleocapsid (N) protein contains two or more RNA binding domains. We investigated the RNA-binding properties of the N-terminal (NTD) and Cterminal (CTD) domain of MHV N. Our results reveal that the NTD specifically interacts with the TRS-L3 sequence. The role of conserved residues (Y127, Y129 and R110) for this specific interaction were systematically investigated. In contrast to the NTD, the MHV CTD is homodimeric in solution and binds single-strand RNA nonspecifically in a binding mode of the noncooperative large ligand lattice model. The CTD dimer binds with a site size, n=4 nucleotide and the appending of the NTD enhances the single-strand nucleic acid binding affinity.