RNA dimerization plays a role in ribosomal frameshifting of the SARS coronavirus

Abstract

Messenger RNA encoded signals that are involved in programmed -1 ribosomal frameshifting (-1 PRF) are typically two-stemmed hairpin (H)-type pseudoknots (pks). We previously described an unusual three-stemmed pseudoknot from the severe acute respiratory syndrome (SARS) coronavirus (CoV) that stimulated -1 PRF. The conserved existence of a third stem–loop suggested an important hitherto unknown function. Here we present new information describing structure and function of the third stem of the SARS pseudoknot. We uncovered RNA dimerization through a palindromic sequence embedded in the SARS-CoV Stem 3. Further in vitro analysis revealed that SARS-CoV RNA dimers assemble through ‘kissing’ loop–loop interactions. We also show that loop–loop kissing complex formation becomes more efficient at physiological temperature and in the presence of magnesium. When the palindromic sequence was mutated, in vitro RNA dimerization was abolished, and frameshifting was reduced from 15 to 5.7%. Furthermore, the inability to dimerize caused by the silent codon change in Stem 3 of SARS-CoV changed the viral growth kinetics and affected the levels of genomic and subgenomic RNA in infected cells. These results suggest that the homodimeric RNA complex formed by the SARS pseudoknot occurs in the cellular environment and that loop–loop kissing interactions involving Stem 3 modulate -1 PRF and play a role in subgenomic and full-length RNA synthesis.

Altering SARS Coronavirus Frameshift Efficiency Affects Genomic and Subgenomic RNA Production

“…The AGU codon was changed to UCC because the latter is present at the same position in the TGEV coronavirus and would be expected to have a minimal impact. More detailed structural information about the L2-UCC mutant is published elsewhere [19]. As expected, a moderate change in frameshifting efficiency was observed (5.72% versus 15%, Figure 3A).…”

The genetic code as expressed through relationships between mRNA structure and protein function

“…3E) [85]. Mutations that interfered with dimerization of this domain, both reduced frameshifting efficiency and inhibited viral replication [85]. These data suggest that the frameshifting efficiency within SARS-CoV is regulated by the ability of the genome to dimerize, connecting two critical steps in the replication cycle of the virus.…”

Multiple Cis-acting elements modulate programmed -1 ribosomal frameshifting in Pea enation mosaic virus

“…It has been proposed that the long-distance interaction is involved in switching between the incompatible activities of RdRp translation and gRNA replication (34), but how the interaction contributes to stimulation of -1PRF/translational readthrough by an RSE remains unexplored. Additional examples of external elements modulating recoding include: (i) a short-distance base-pairing interaction between two stem-loops downstream of the slippery site that is required for -1PRF of bacterial transposable elements in the IS51 group of the IS3 family (36); and (ii) an intermolecular kissing loop-loop interaction in SARS-CoV that stimulates -1PRF through dimerization of the viral genomic (g)RNA (37). This growing number of critical base-pairing interactions between RSE and non-continuous elements suggests a general necessity for such interactions to precisely control recoding in gRNA.…”