From Recoding to Peptides for MHC Class I Immune Display: Enriching Viral Expression, Virus Vulnerability and Virus Evasion

Abstract

Many viruses, especially RNA viruses, utilize programmed ribosomal frameshifting and/or stop codon readthrough in their expression, and in the decoding of a few a UGA is dynamically redefined to specify selenocysteine. This recoding can effectively increase viral coding capacity and generate a set ratio of products with the same N-terminal domain(s) but different C-terminal domains. Recoding can also be regulatory or generate a product with the non-universal 21st directly encoded amino acid. Selection for translation speed in the expression of many viruses at the expense of fidelity creates host immune defensive opportunities. In contrast to host opportunism, certain viruses, including some persistent viruses, utilize recoding or adventitious frameshifting as part of their strategy to evade an immune response or specific drugs. Several instances of recoding in small intensively studied viruses escaped detection for many years and their identification resolved dilemmas. The fundamental importance of ribosome ratcheting is consistent with the initial strong view of invariant triplet decoding which however did not foresee the possibility of transitory anticodon:codon dissociation. Deep level dynamics and structural understanding of recoding is underway, and a high level structure relevant to the frameshifting required for expression of the SARS CoV-2 genome has just been determined.

Keywords: DRiPs; StopGo; bet hedging; cancer; codon redefinition; ribosomal frameshifting; ribosome structure; selenocysteine; stop codon readthrough.

Figure 1

Outreach representations of frameshifting and readthrough. Panel (a) depicts retroviral recoding where protease sequencing was important for mechanistic understanding: top, Murine Leukemia Virus gag stop codon Readthrough; bottom, Mouse Mammary Tumor Virus gag pol Frameshifting. The head motifs used to depict directionality were inspired by the 1200-year old ‘Book of Kells’ in Trinity College Dublin library. The embedded ribosomes have their A, P, and E sites in green and those shown on the left are at an earlier stage of decoding than those on the right. Correspondingly, on the left the proportion of the mRNA (in red) that has passed through the ribosomes is small in contrast to that shown on the right side, and the nascent peptide emerging from the ribosome (blue) is longer on the right side than on the left. Gag is represented in ochre and Pol in green. These images are from a band of recoding tiles positioned around the middle of the outside walls of ‘a house’ in S.W. Cork, Ireland. Panel (b) shows the decoding seat component of the sculpture in the National Botanic Gardens, Dublin entitled ‘What is Life’. The title follows that used by Erwin Schrödinger of the Dublin Institute of Advanced Studies for his 1944 book (and previous year lectures). Both Watson and Crick independently credited the book ‘What is Life?’ as an early source of inspiration for them. A description of the components of the sculpture, including a hammerhead ribozyme and a ribosome can be found at http://whatislife.ie/ (accessed on 1 May 2021). The decoding seat is on a mound overlooking an iconic Charles Jencks 5.5 m high DNA double helix similar to those at Clare College Cambridge and near Cold Spring Harbor Laboratory beach. Each seat panel represents a codon. Three bars below and above each panel reflect its 3nt composition. Starting from the left, or 5′ end, the initial panels reflect all zero frame reading. A proportion of ribosomes shifting to the -1 frame is represented by the first split panel in which part of the panel is offset to the left by one third of a panel length. Continued triplet decoding by frameshifted ribosomes, and by zero frame ribosomes is represented by the panel at the right end.

Figure 2

SARS-CoV-2 genomic (gRNA) and subgenomic RNA (sgRNA) structures. −1 FS indicates the site of −1 frameshifting.

Figure 3

(A) Schematic of secondary structure elements regulating -1 frameshifting in SARS-CoV-2. As visualized by cryo-EM [98], the frameshift stimulatory pseudoknot consists of three stems, interconnected by unstructured single-stranded loops. (B) Visualization of the stimulatory pseudknot bound to an elongating ribosome at the vicinity of the frameshift site. The intact structured pseudoknot is present at the entry of the mRNA channel on the ribosomal small subunit (SSU). SSU proteins are colored in yellow, LSU proteins in blue, and rRNA in gray. The pseudoknot is colored as per secondary structure description in panel A. (C) Pseudoknot as observed from the solvent exposed side of the SSU. Stems 1 and 2 are quasi co-axially stacked, with Stem 3 being perpendicular. Stem 1 interacts with helix 16 of 18s rRNA, potentially aiding the pseudoknot in restricting ribosomal translocation. These figures were adapted from Bhatt et al. [98].

Figure 4

(A) Frameshifting in EMCV (and TMEV) is stimulated by a protein:RNA complex positioned at the leading edge of the ribosome when the decoding center is on the shift site. The viral encoded 2A protein, which is encoded 5′ of the shift site, binds to the 3‘ stem loop to stimulate frameshifting. (B) Frameshifting in PRRSV is stimulated by a protein:RNA complex that requires dimerization of the virally encoded nsp1B protein and the chromosomally encoded polyC binding protein which bind to the 3‘ stem loop to stimulate frameshifting.

Figure 5

Cartoon structure of merafloxacin [165], an inhibitor of SARS-CoV-2 frameshifting [98,165].

Figure 6

Ribosomal proteins that impact MHC Class 1 peptide generation for immunosurveillance, according to Wei et al. 2019 [192]. (a–c) Depiction of ribosomal proteins that impact MHC Class 1 peptide generation for immunosurveillance [192]. rpL6 and rpL28, which are adjacent to each other on the large subunit, have opposing effects on viral peptide generation. rpL6 depletion decreases ubiquitin-dependent peptide presentation, whereas rpL28 depletion increases ubiquitin-dependent and -independent peptide presentation. Figure was generated with a human ribosome (PDB 4V6X) in ChimeraX. (d) Individual ribosomal protein knockdowns affect immunosurveillance by impacting either MHC Class 1 surface expression or viral peptide generation, and are grouped as reported.

Figure 7

Map of KSHV LANA1 and EBV EBNA1 and amino acid sequence alignment comparing LANA1.CR2 and EBNA1.GAr in the −2 frame (EBNA1ARF). EBNA1 comprises the N terminus, GA-rich central domain, and C-terminal DNA binding domain. Stop codons of the −2 frame are indicated with arrows. Although the EBNA1 GAr (0 frame) has no amino acid similarity to the LANA1 CR, EBNA1ARF has ∼35% similarity to the 0 frame of the LANA1 CR2 domain, and both sequences contain highly acidic QE-rich repeats. From Ref. [5].