Achieving a Golden Mean: Mechanisms by Which Coronaviruses Ensure Synthesis of the Correct Stoichiometric Ratios of Viral Proteins

Laboratory of Hepatitis and Related Emerging Agents, Division of Emerging and Transfusion-Transmitted Diseases, Office of Blood Research and Review, CBER, FDA, 8800 Rockville Pike, HFM310, Bethesda, Maryland 20892, USA.
Journal of Virology (Impact Factor: 4.44). 02/2010; 84(9):4330-40. DOI: 10.1128/JVI.02480-09
Source: PubMed


In retroviruses and the double-stranded RNA totiviruses, the efficiency of programmed -1 ribosomal frameshifting is critical for ensuring the proper ratios of upstream-encoded capsid proteins to downstream-encoded replicase enzymes. The genomic organizations of many other frameshifting viruses, including the coronaviruses, are very different, in that their upstream open reading frames encode nonstructural proteins, the frameshift-dependent downstream open reading frames encode enzymes involved in transcription and replication, and their structural proteins are encoded by subgenomic mRNAs. The biological significance of frameshifting efficiency and how the relative ratios of proteins encoded by the upstream and downstream open reading frames affect virus propagation has not been explored before. Here, three different strategies were employed to test the hypothesis that the -1 PRF signals of coronaviruses have evolved to produce the correct ratios of upstream- to downstream-encoded proteins. Specifically, infectious clones of the severe acute respiratory syndrome (SARS)-associated coronavirus harboring mutations that lower frameshift efficiency decreased infectivity by >4 orders of magnitude. Second, a series of frameshift-promoting mRNA pseudoknot mutants was employed to demonstrate that the frameshift signals of the SARS-associated coronavirus and mouse hepatitis virus have evolved to promote optimal frameshift efficiencies. Finally, we show that a previously described frameshift attenuator element does not actually affect frameshifting per se but rather serves to limit the fraction of ribosomes available for frameshifting. The findings of these analyses all support a "golden mean" model in which viruses use both programmed ribosomal frameshifting and translational attenuation to control the relative ratios of their encoded proteins.

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    • "These revelations should be of much interest when further studies on the programmed frameshifting mechanism are being planned. By contrast, a much more stable structure, such as the original ATT in SARS CoV, could still affect viral −1 PRF efficiency by serving as a translational attenuator as previously proposed [15]. However, deletion of six nucleotides to disrupt the minimal upstream hairpin stem in an ATT-containing construct (construct 13318–13520 Δ6 in Fig. 4B) restored −1 PRF efficiency to that of the ATT-lacking 13390–13520 construct in vitro (compare lanes 1 and 4 of Fig. 4B and Fig. 1B). "
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    ABSTRACT: RNA structures are unwound for decoding. In the process, they can pause the elongating ribosome for regulation. An example is the stimulation of -1 programmed ribosomal frameshifting, leading to 3' direction slippage of the reading-frame during elongation, by specific pseudoknot stimulators downstream of the frameshifting site. By investigating a recently identified regulatory element upstream of the SARS coronavirus (SARS-CoV) -1 frameshifting site, it is shown that a minimal functional element with hairpin forming potential is sufficient to down-regulate-1 frameshifting activity. Mutagenesis to disrupt or restore base pairs in the potential hairpin stem reveals that base-pair formation is required for-1 frameshifting attenuation in vitro and in 293T cells. The attenuation efficiency of a hairpin is determined by its stability and proximity to the frameshifting site; however, it is insensitive to E site sequence variation. Additionally, using a dual luciferase assay, it can be shown that a hairpin stimulated +1 frameshifting when placed upstream of a +1 shifty site in yeast. The investigations indicate that the hairpin is indeed a cis-acting programmed reading-frame switch modulator. This result provides insight into mechanisms governing-1 frameshifting stimulation and attenuation. Since the upstream hairpin is unwound (by a marching ribosome) before the downstream stimulator, this study's findings suggest a new mode of translational regulation that is mediated by the reformed stem of a ribosomal unwound RNA hairpin during elongation.
    PLoS ONE 04/2013; 8(4):e62283. DOI:10.1371/journal.pone.0062283 · 3.23 Impact Factor
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    • "To determine the importance of L2-mediated self-association for SARS-CoV -1 PRF, several pseudoknot variants were subjected to a dual luciferase reporter-based frameshifting analysis as previously described (Figure 8A) (1,39). A Loop 2 ACUucc silent (serine codon) mutation was made to investigate the role of the palindrome (S3L2-ACUucc; Figure 1D) in promoting -1 PRF. "
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    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.
    Nucleic Acids Research 12/2012; 41(4). DOI:10.1093/nar/gks1361 · 9.11 Impact Factor
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    • "Multiple models have been proposed to explain the frameshift mechanism (1,6,31–38). Common among them are the following steps: (i) during translation, the ribosome pauses when the slippery sequence (UUU UUA in the 0 frame) is engaged in the ribosomal A- and P-sites (4,18,22,39). "
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    ABSTRACT: The human immunodeficiency virus (HIV) requires a programmed -1 ribosomal frameshift for Pol gene expression. The HIV frameshift site consists of a heptanucleotide slippery sequence (UUUUUUA) followed by a spacer region and a downstream RNA stem-loop structure. Here we investigate the role of the RNA structure in promoting the -1 frameshift. The stem-loop was systematically altered to decouple the contributions of local and overall thermodynamic stability towards frameshift efficiency. No correlation between overall stability and frameshift efficiency is observed. In contrast, there is a strong correlation between frameshift efficiency and the local thermodynamic stability of the first 3-4 bp in the stem-loop, which are predicted to reside at the opening of the mRNA entrance channel when the ribosome is paused at the slippery site. Insertion or deletions in the spacer region appear to correspondingly change the identity of the base pairs encountered 8 nt downstream of the slippery site. Finally, the role of the surrounding genomic secondary structure was investigated and found to have a modest impact on frameshift efficiency, consistent with the hypothesis that the genomic secondary structure attenuates frameshifting by affecting the overall rate of translation.
    Nucleic Acids Research 12/2012; 41(3). DOI:10.1093/nar/gks1254 · 9.11 Impact Factor
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