Structural and Functional Insights into Viral Programmed Ribosomal Frameshifting

Figures

• 

  Figure 1. Schematic diagram summarizing the molecules and principles governing viral −1PRF. The eukaryotic ribosome is depicted in the act of translating a frameshift-stimulatory signal in an mRNA. Key players with frameshift-modulatory activity are labelled. Abbreviations: −1PRF, programmed −1 ribosomal frameshifting; A, aminoacyl-site; mRNA, messenger RNA; ORF, open reading frame; P, peptidyl-site; tRNA, transfer RNA.

• 

  Figure 2. Anticodon loop modifications associated with −1PRF. A schematic of a tRNA is shown on the left, with a zoom-in of the anticodon loop on the right. Modified wobble-bases include 5-mcm5s2U and Q. The modified base yW is found adjacent to the anticodon (residues 34–36). Abbreviations: −1PRF, programmed −1 ribosomal frameshifting; mcm5s2U, 5-methoxycarbonylmethyl-2-thiouridine; Q, queuosine; tRNA, transfer RNA; yW, wybutosine.

• 

  Figure 3. Illustrating the structural diversity of −1PRF stimulatory elements in canonical (a–d) and protein-stimulated (e,f) frameshifting. The upper part of each panel shows a summary of the viral translation products. The genomic frame of each protein relative to the main ORF (0; blue) is indicated. In addition to PRF, products encoded in other reading frames may be accessed by alternative splicing, translation of subgenomic RNAs, leaky scanning, or stop codon read through. The lower part of each panel shows a topological diagram and representative 3D structure of stimulatory elements, color coded by feature. In panel e, the proposed arrangement of components is informed by biochemical experiments and docking into a SAXS envelope (gray). In panel f, the structure of the 2A protein is known but the tertiary structure of the cognate RNA element is unknown. Abbreviations: −1PRF, programmed −1 ribosomal frameshifting; BWYV, beet western yellows virus; EMCV, encephalomyocarditis virus; FS, frameshift site; HIV-1, human immunodeficiency virus 1; MMTV, mouse mammary tumor virus; mRNA, messenger RNA; nsp, nonstructural protein; ORF, open reading frame; PCBP, poly(C) binding protein; PRRSV,…

• 

  Figure 4. Illustrating the conformational repertoire of two −1PRF stimulatory elements. (a) Topological diagram and structures of SARS-CoV-2 pseudoknots, color coded by feature. PDB codes are indicated above each structure. (inset) Example of 5′ end threading through the S1/S3/L3 junction. Backbone alignment by least-squares superposition reveals the principal modes of flexibility. (b) As in panel a but for HIV-1 stem-loops. Abbreviations: −1PRF, programmed −1 ribosomal frameshifting; HIV-1, human immunodeficiency virus 1; PDB, Protein Data Bank; SARS, severe acute respiratory syndrome; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

• 

  Figure 5. Schematic of the elongation cycle of translation. The steps and intermediates shown are based on prokaryotic translation, with equivalent eukaryotic factors shown in brackets. Abbreviations: A, aminoacyl site; E, exit site; eEF1A, eukaryotic elongation factor 1A; eEF2, eukaryotic elongation factor 2; EF-G, elongation factor G; EF-Tu, elongation factor Tu; P, peptidyl site; T, EF-Tu-/eEF1A-tRNA binding site.

• 

  Figure 6. Interactions between the ribosome and −1PRF stimulatory elements. The large ribosomal subunit is omitted for clarity. (a) 80S ribosomes stalled one codon before the slippery sequence in a preframeshift complex with the SARS-CoV-2 pseudoknot. The P-site tRNA (dark gray), mRNA (light gray), and pseudoknot (colored as in previous figures) are shown. (inset) Zoomed-in views showing interactions between the SARS-CoV-2 pseudoknot, ribosome helicase subunits uS3 and eS10, and 18S rRNA helix 16. (b) Reconstituted Escherichia coli 70S ribosomes at the HIV-1 frameshift site. The stem-loop can fold within the A-site, preventing tRNA delivery. (inset) Spacer and GGA bulge stack against base of upper stem in the A-site. In the P/E rotated state, the stem-loop rotates by ~15° and contacts helix 38 from the large subunit. (c) Reconstituted Thermus thermophilus 70S ribosomes at the bacterial dnaX frameshift site. The stem-loop stimulatory element is visible at the ribosome helicase. (inset) The stem-loop is in close contact with ribosome helicase subunit uS3. (d) Reconstituted E. coli 70S ribosomes in complex with EMCV 2A. (inset) 2A binds to 16S rRNA at the GTPase binding site, potentially…

• 

  Figure 7. Small-molecule binding sites on the HIV-1 stem-loop, targeting the upper helix. Compounds are shown as sticks in the zoomed-in views. Abbreviation: HIV-1, human immunodeficiency virus 1.