High-resolution structures reveal that yeast ribosomal protein L11 and its bacterial/archael homologs called L5 contain a highly conserved, basically charged internal loop that interacts with the peptidyl-transfer RNA (tRNA) T-loop. We call this the L11 ‘P-site loop’. Chemical protection of wild-type ribosome shows that that the P-site loop is inherently flexible, i.e. it is extended into the ribosomal P-site when this is unoccupied by tRNA, while it is retracted into the terminal loop of 25S rRNA Helix 84 when the P-site is occupied. To further analyze the function of this structure, a series of mutants within the P-site loop were created and analyzed. A mutant that favors interaction of the P-site loop with the terminal loop of Helix 84 promoted increased affinity for peptidyl-tRNA, while another that favors its extension into the ribosomal P-site had the opposite effect. The two mutants also had opposing effects on binding of aa-tRNA to the ribosomal A-site, and downstream functional effects were observed on translational fidelity, drug resistance/hypersensitivity, virus maintenance and overall cell growth. These analyses suggest that the L11 P-site loop normally helps to optimize ribosome function by monitoring the occupancy status of the ribosomal P-site.

Yeast ribosomal proteins L11 and S18 form a dynamic intersubunit interaction called the B1b/c bridge. Recent high resolution images of the ribosome have enabled targeting of specific residues in this bridge to address how distantly separated regions within the large and small subunits of the ribosome communicate with each other. Mutations were generated in the L11 side of the B1b/c bridge with a particular focus on disrupting the opposing charge motifs that have previously been proposed to be involved in subunit ratcheting. Mutants had wide-ranging effects on cellular viability and translational fidelity, with the most pronounced phenotypes corresponding to amino acid changes resulting in alterations of local charge properties. Chemical protection studies of selected mutants revealed rRNA structural changes in both the large and small subunits. In the large subunit rRNA, structural changes mapped to Helices 39, 80, 82, 83, 84, and the peptidyltransferase center. In the small subunit rRNA, structural changes were identified in helices 30 and 42, located between S18 and the decoding center. The rRNA structural changes correlated with charge-specific alterations to the L11 side of the B1b/c bridge. These analyses underscore the importance of the opposing charge mechanism in mediating B1b/c bridge interactions and suggest an extensive network of information exchange between distinct regions of the large and small subunits.

EDP-938 is a novel non-fusion replication inhibitor of respiratory syncytial virus (RSV). It is highly active against all RSV-A and B laboratory strains and clinical isolates tested in vitro in various cell lines and assays, with half-maximal effective concentrations (EC50s) of 21, 23 and 64 nM against Long (A), M37 (A) and VR-955 (B) strains, respectively, in the primary human bronchial epithelial cells (HBECs). EDP-938 inhibits RSV at a post-entry replication step of the viral life cycle as confirmed by time-of-addition study, and the activity appears to be mediated by viral nucleoprotein (N). In vitro resistance studies suggest that EDP-938 presents a higher barrier to resistance compared to viral fusion or non-nucleoside L polymerase inhibitors with no cross-resistance observed. Combinations of EDP-938 with other classes of RSV inhibitors lead to synergistic antiviral activity in vitro. Finally, EDP-938 has also been shown to be efficacious in vivo in a non-human primate model of RSV infection.

Programmed −1 ribosomal frameshifting (−1 PRF) is a mechanism that directs elongating ribosomes to shift-reading frame by 1 base in the 5′ direction that is utilized by many RNA viruses. Importantly, rates of −1 PRF are fine-tuned by viruses, including Retroviruses, Coronaviruses, Flavivriuses and in two endogenous viruses of the yeast Saccharomyces cerevisiae, to deliver the correct ratios of different viral proteins for efficient replication. Thus, −1 PRF presents a novel target for antiviral therapeutics. The underlying molecular mechanism of −1 PRF is conserved from yeast to mammals, enabling yeast to be used as a logical platform for high-throughput screens. Our understanding of the strengths and pitfalls of assays to monitor −1 PRF have evolved since the initial discovery of −1 PRF. These include controlling for the effects of drugs on protein expression and mRNA stability, as well as minimizing costs and the requirement for multiple processing steps. Here we describe the development of an automated yeast-based dual fluorescence assay of −1 PRF that provides a rapid, inexpensive automated pipeline to screen for compounds that alter rates of −1 PRF which will help to pave the way toward the discovery and development of novel antiviral therapeutics.