Abstract
Viral −1 programmed ribosomal frameshifting (PRF) as a potential antiviral target has attracted interest because many human viral pathogens, including human immunodeficiency virus (HIV) and coronaviruses, rely on −1 PRF for optimal propagation. Efficient eukaryotic −1 PRF requires an optimally placed stimulator structure downstream of the frameshifting site and different strategies targeting viral −1 PRF stimulators have been developed. However, accessing particular −1 PRF stimulator information represents a bottle-neck in combating the emerging epidemic viral pathogens such as Middle East respiratory syndrome coronavirus (MERS-CoV). Recently, an RNA hairpin upstream of frameshifting site was shown to act as a cis-element to attenuate −1 PRF with mechanism unknown. Here, we show that an upstream duplex formed in-trans, by annealing an antisense to its complementary mRNA sequence upstream of frameshifting site, can replace an upstream hairpin to attenuate −1 PRF efficiently. This finding indicates that the formation of a proximal upstream duplex is the main determining factor responsible for −1 PRF attenuation and provides mechanistic insight. Additionally, the antisense-mediated upstream duplex approach downregulates −1 PRF stimulated by distinct −1 PRF stimulators, including those of MERS-CoV, suggesting its general application potential as a robust means to evaluating viral −1 PRF inhibition as soon as the sequence information of an emerging human coronavirus is available.
Highlights
Reading-frame maintenance is crucial for translational fidelity because it ensures that codons are in the correct reading-frame of an mRNA on delivery into the A site of an elongating ribosome
Value for each bar is the mean of three independent experiments with standard error of the mean
A frameshifting pseudoknot can prohibit a significant fraction of frameshifted ribosomes that it stimulated from reaching the −1 frame stop codon. It leads to the compromise of observed −1 programmed reading-frame shift (PRF) efficiency [49]
Summary
Reading-frame maintenance is crucial for translational fidelity because it ensures that codons are in the correct reading-frame of an mRNA on delivery into the A site of an elongating ribosome. Efficient eukaryotic −1 PRF requires two cis-acting elements in mRNA, a slippery sequence (where frameshifting occurs) and an optimally placed downstream stimulator structure. An X XXY YYZ sequence in the slippery site facilitates −1 frameshifting by paving codon-anticodon disruption in the P and A sites of the 0-frame (XXY and YYZ codons) and codon-anticodon repairing in the −1 frame (XXX and YYY codons) This transition is further enhanced by resistance from the downstream stimulator (usually a pseudoknot or a hairpin) to the duplex unwinding activity of ribosome, leading to interference in the translocation step of an elongation cycle [9,10,11,12,13].
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