scholarly journals Ribosomal pausing during translation of an RNA pseudoknot.

1993 ◽  
Vol 13 (11) ◽  
pp. 6931-6940 ◽  
Author(s):  
P Somogyi ◽  
A J Jenner ◽  
I Brierley ◽  
S C Inglis
Keyword(s):  
High Efficiency ◽  
Mutational Analysis ◽  
Base Pairs ◽  
Stem Loop ◽  
Pseudoknot Structure ◽  
Stem Loop Structure ◽  
Dead End ◽  
Rna Pseudoknot ◽  
Slippery Sequence

The genomic RNA of the coronavirus infectious bronchitis virus contains an efficient ribosomal frameshift signal which comprises a heptanucleotide slippery sequence followed by an RNA pseudoknot structure. The presence of the pseudoknot is essential for high-efficiency frameshifting, and it has been suggested that its function may be to slow or stall the ribosome in the vicinity of the slippery sequence. To test this possibility, we have studied translational elongation in vitro on mRNAs engineered to contain a well-defined pseudoknot-forming sequence. Insertion of the pseudoknot at a specific location within the influenza virus PB1 mRNA resulted in the production of a new translational intermediate corresponding to the size expected for ribosomal arrest at the pseudoknot. The appearance of this protein was transient, indicating that it was a true paused intermediate rather than a dead-end product, and mutational analysis confirmed that its appearance was dependent on the presence of a pseudoknot structure within the mRNA. These observations raise the possibility that a pause is required for the frameshift process. The extent of pausing at the pseudoknot was compared with that observed at a sequence designed to form a simple stem-loop structure with the same base pairs as the pseudoknot. This structure proved to be a less effective barrier to the elongating ribosome than the pseudoknot and in addition was unable to direct efficient ribosomal frameshifting, as would be expected if pausing plays an important role in frameshifting. However, the stem-loop was still able to induce significant pausing, and so this effect alone may be insufficient to account for the contribution of the pseudoknot to frameshifting.

scholarly journals Ribosomal Pausing at a Frameshifter RNA Pseudoknot Is Sensitive to Reading Phase but Shows Little Correlation with Frameshift Efficiency

2001 ◽  
Vol 21 (24) ◽  
pp. 8657-8670 ◽  
Author(s):  
Harry Kontos ◽  
Sawsan Napthine ◽  
Ian Brierley
Keyword(s):  
Rna Structure ◽  
Phase Dependence ◽  
Stem Loop ◽  
Reading Frame ◽  
Stem Loop Structure ◽  
Rna Pseudoknot ◽  
Ribosomal P ◽  
Slippery Sequence ◽  
The Impact ◽  
Ribosomal Pausing

ABSTRACT Here we investigated ribosomal pausing at sites of programmed −1 ribosomal frameshifting, using translational elongation and ribosome heelprint assays. The site of pausing at the frameshift signal of infectious bronchitis virus (IBV) was determined and was consistent with an RNA pseudoknot-induced pause that placed the ribosomal P- and A-sites over the slippery sequence. Similarly, pausing at the simian retrovirus 1 gag/pol signal, which contains a different kind of frameshifter pseudoknot, also placed the ribosome over the slippery sequence, supporting a role for pausing in frameshifting. However, a simple correlation between pausing and frameshifting was lacking. Firstly, a stem-loop structure closely related to the IBV pseudoknot, although unable to stimulate efficient frameshifting, paused ribosomes to a similar extent and at the same place on the mRNA as a parental pseudoknot. Secondly, an identical pausing pattern was induced by two pseudoknots differing only by a single loop 2 nucleotide yet with different functionalities in frameshifting. The final observation arose from an assessment of the impact of reading phase on pausing. Given that ribosomes advance in triplet fashion, we tested whether the reading frame in which ribosomes encounter an RNA structure (the reading phase) would influence pausing. We found that the reading phase did influence pausing but unexpectedly, the mRNA with the pseudoknot in the phase which gave the least pausing was found to promote frameshifting more efficiently than the other variants. Overall, these experiments support the view that pausing alone is insufficient to mediate frameshifting and additional events are required. The phase dependence of pausing may be indicative of an activity in the ribosome that requires an optimal contact with mRNA secondary structures for efficient unwinding.

scholarly journals Characterization of RNA Elements That Regulate Gag-Pol Ribosomal Frameshifting in Equine Infectious Anemia Virus

2003 ◽  
Vol 77 (19) ◽  
pp. 10280-10287 ◽  
Author(s):  
Chaoping Chen ◽  
Ronald C. Montelaro
Keyword(s):  
Viral Replication ◽  
Rna Structure ◽  
Equine Infectious Anemia Virus ◽  
Stop Codon ◽  
Structural Determinants ◽  
Ribosomal Frameshifting ◽  
Reading Frame ◽  
Anemia Virus ◽  
Antiviral Targets ◽  
Slippery Sequence

ABSTRACT Synthesis of Gag-Pol polyproteins of retroviruses requires ribosomes to shift translational reading frame once or twice in a −1 direction to read through the stop codon in the gag reading frame. It is generally believed that a slippery sequence and a downstream RNA structure are required for the programmed −1 ribosomal frameshifting. However, the mechanism regulating the Gag-Pol frameshifting remains poorly understood. In this report, we have defined specific mRNA elements required for sufficient ribosomal frameshifting in equine anemia infectious virus (EIAV) by using full-length provirus replication and Gag/Gag-Pol expression systems. The results of these studies revealed that frameshifting efficiency and viral replication were dependent on a characteristic slippery sequence, a five-base-paired GC stretch, and a pseudoknot structure. Heterologous slippery sequences from human immunodeficiency virus type 1 and visna virus were able to substitute for the EIAV slippery sequence in supporting EIAV replication. Disruption of the GC-paired stretch abolished the frameshifting required for viral replication, and disruption of the pseudoknot reduced the frameshifting efficiency by 60%. Our data indicated that maintenance of the essential RNA signals (slippery sequences and structural elements) in this region of the genomic mRNA was critical for sufficient ribosomal frameshifting and EIAV replication, while concomitant alterations in the amino acids translated from the same region of the mRNA could be tolerated during replication. The data further indicated that proviral mutations that reduced frameshifting efficiency by as much as 50% continued to sustain viral replication and that greater reductions in frameshifting efficiency lead to replication defects. These studies define for the first time the RNA sequence and structural determinants of Gag-Pol frameshifting necessary for EIAV replication, reveal novel aspects relative to frameshifting elements described for other retroviruses, and provide new genetic determinants that can be evaluated as potential antiviral targets.

scholarly journals Two Nucleotides Immediately Upstream of the Essential A6G3 Slippery Sequence Modulate the Pattern of G Insertions during Sendai Virus mRNA Editing

1999 ◽  
Vol 73 (1) ◽  
pp. 343-351 ◽  
Author(s):  
Stéphane Hausmann ◽  
Dominique Garcin ◽  
Anne-Sophie Morel ◽  
Daniel Kolakofsky
Keyword(s):  
Sendai Virus ◽  
Cell System ◽  
Mrna Editing ◽  
Reading Frame ◽  
Recombinant Viruses ◽  
P Gene ◽  
Minimum Number ◽  
Type 3 ◽  
Slippery Sequence ◽  
Bovine Parainfluenza Virus

ABSTRACT Editing of paramyxovirus P gene mRNAs occurs cotranscriptionally and functions to fuse an alternate downstream open reading frame to the N-terminal half of the P protein. G residues are inserted into a short G run contained within a larger purine run (A n G n ) in this process, by a mechanism whereby the transcribing polymerase stutters (i.e., reads the same template cytosine more than once). Although Sendai virus (SeV) and bovine parainfluenza virus type 3 (bPIV3) are closely related, the G insertions in their P mRNAs are distributed differently. SeV predominantly inserts a single G residue within the G run of the sequence 5′ AACAAAAAAGGG, whereas bPIV3 inserts one to six G’s at roughly equal frequency within the sequence 5′ AUUAAAAAAGGGG(differences are underlined). We have examined how thecis-acting editing sequence determines the number of G’s inserted, both in a transfected cell system using minigenome analogues and by generating recombinant viruses. We found that the presence of four rather than three G’s in the purine run did not affect the distribution of G insertions. However, when the underlined AC of the SeV sequence was replaced by the UU found in bPIV3, the editing phenotype from both the minigenome and the recombinant virus resembled that found in natural bPIV3 infections (i.e., a significant fraction of the mRNAs contained two to six G insertions). The two nucleotides located just upstream of the polypurine tract are thus key determinants of the editing phenotype of these viruses. Moreover, the minimum number of A residues that will promote SeV editing phenotype is six but can be reduced to five when the upstream AC is replaced by UU. A model for how the upstream dinucleotide controls the insertion phenotype is presented.

scholarly journals Programmed −2/−1 Ribosomal Frameshifting in Simarteriviruses: an Evolutionarily Conserved Mechanism

2019 ◽  
Vol 93 (16) ◽  
Author(s):  
Yanhua Li ◽  
Andrew E. Firth ◽  
Ian Brierley ◽  
Yingyun Cai ◽  
Sawsan Napthine ◽  
...  
Keyword(s):  
Nonhuman Primates ◽  
Hemorrhagic Fever ◽  
Viral Proteins ◽  
Equine Arteritis Virus ◽  
Virus Growth ◽  
Ribosomal Frameshifting ◽  
Simian Hemorrhagic Fever Virus ◽  
Evolutionarily Conserved ◽  
Slippery Sequence

ABSTRACTThe −2/−1 programmed ribosomal frameshifting (−2/−1 PRF) mechanism in porcine reproductive and respiratory syndrome virus (PRRSV) leads to the translation of two additional viral proteins, nonstructural protein 2TF (nsp2TF) and nsp2N. This −2/−1 PRF mechanism is transactivated by a viral protein, nsp1β, and cellular poly(rC) binding proteins (PCBPs). Critical elements for −2/−1 PRF, including a slippery sequence and a downstream C-rich motif, were also identified in 11 simarteriviruses. However, the slippery sequences (XXXUCUCU instead of XXXUUUUU) in seven simarteriviruses can only facilitate −2 PRF to generate nsp2TF. The nsp1β of simian hemorrhagic fever virus (SHFV) was identified as a key factor that transactivates both −2 and −1 PRF, and the universally conserved Tyr111 and Arg114 in nsp1β are essential for this activity.In vitrotranslation experiments demonstrated the involvement of PCBPs in simarterivirus −2/−1 PRF. Using SHFV reverse genetics, we confirmed critical roles of nsp1β, slippery sequence, and C-rich motif in −2/−1 PRF in SHFV-infected cells. Attenuated virus growth ability was observed in SHFV mutants with impaired expression of nsp2TF and nsp2N. Comparative genomic sequence analysis showed that key elements of −2/−1 PRF are highly conserved in all known arteriviruses except equine arteritis virus (EAV) and wobbly possum disease virus (WPDV). Furthermore, −2/−1 PRF with SHFV PRF signal RNA can be stimulated by heterotypic nsp1βs of all non-EAV arteriviruses tested. Taken together, these data suggest that −2/−1 PRF is an evolutionarily conserved mechanism employed in non-EAV/-WPDV arteriviruses for the expression of additional viral proteins that are important for viral replication.IMPORTANCESimarteriviruses are a group of arteriviruses infecting nonhuman primates, and a number of new species have been established in recent years. Although these arteriviruses are widely distributed among African nonhuman primates of different species, and some of them cause lethal hemorrhagic fever disease, this group of viruses has been undercharacterized. Since wild nonhuman primates are historically important sources or reservoirs of human pathogens, there is concern that simarteriviruses may be preemergent zoonotic pathogens. Thus, molecular characterization of simarteriviruses is becoming a priority in arterivirology. In this study, we demonstrated that an evolutionarily conserved ribosomal frameshifting mechanism is used by simarteriviruses and other distantly related arteriviruses for the expression of additional viral proteins. This mechanism is unprecedented in eukaryotic systems. Given the crucial role of ribosome function in all living systems, the potential impact of the in-depth characterization of this novel mechanism reaches beyond the field of virology.

scholarly journals Structure–function analysis of the ribosomal frameshifting signal of two human immunodeficiency virus type 1 isolates with increased resistance to viral protease inhibitors

2007 ◽  
Vol 88 (1) ◽  
pp. 226-235 ◽  
Author(s):  
Roseanne Girnary ◽  
Louise King ◽  
Laurence Robinson ◽  
Robert Elston ◽  
Ian Brierley
Keyword(s):  
Human Immunodeficiency Virus ◽  
Protease Inhibitors ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Ribosomal Frameshift ◽  
Viral Protease ◽  
Immunodeficiency Virus ◽  
Slippery Sequence ◽  
Hiv 1

Expression of the pol-encoded proteins of human immunodeficiency virus type 1 (HIV-1) requires a programmed –1 ribosomal frameshift at the junction of the gag and pol coding sequences. Frameshifting takes place at a heptanucleotide slippery sequence, UUUUUUA, and is enhanced by a stimulatory RNA structure located immediately downstream. In patients undergoing viral protease (PR) inhibitor therapy, a p1/p6gag L449F cleavage site (CS) mutation is often observed in resistant isolates and frequently generates, at the nucleotide sequence level, a homopolymeric and potentially slippery sequence (UUUUCUU to UUUUUUU). The mutation is located within the stimulatory RNA downstream of the authentic slippery sequence and could act to augment levels of pol-encoded enzymes to counteract the PR deficit. Here, RNA secondary structure probing was employed to investigate the structure of a CS-containing frameshift signal, and the effect of this mutation on ribosomal frameshift efficiency in vitro and in tissue culture cells was determined. A second mutation, a GGG insertion in the loop of the stimulatory RNA that could conceivably lead to resistance by enhancing the activity of the structure, was also tested. It was found, however, that the CS and GGG mutations had only a very modest effect on the structure and activity of the HIV-1 frameshift signal. Thus the increased resistance to viral protease inhibitors seen with HIV-1 isolates containing mutations in the frameshifting signal is unlikely to be accounted for solely by enhancement of frameshift efficiency.

scholarly journals Stepwise Evolution and Exceptional Conservation of ORF1a/b Overlap in Coronaviruses

2021 ◽  
Author(s):  
Han Mei ◽  
Sergei Kosakovsky Pond ◽  
Anton Nekrutenko
Keyword(s):  
Comparative Analysis ◽  
Secondary Structure ◽  
Nucleotide Substitution ◽  
Genomic Data ◽  
Signatures Of Selection ◽  
Slippery Sequence ◽  
Stepwise Evolution ◽  
Reading Frames

Abstract The programmed frameshift element (PFE) rerouting translation from ORF1a to ORF1b is essential for propagation of coronaviruses. The overlap between the two reading frames, a slippery sequence, and an ensemble of secondary structure elements places severe constraints on this region as most possible nucleotide substitution may disrupt one or more of these features. Here we performed a comparative analysis of all available coronaviral genomic data available to date to demonstrate exceptional conservation and detect signatures of selection within the PFE region.

scholarly journals Thermodynamic control of −1 programmed ribosomal frameshifting

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Lars V. Bock ◽  
Neva Caliskan ◽  
Natalia Korniy ◽  
Frank Peske ◽  
Marina V. Rodnina ◽  
...  
Keyword(s):  
Energy Difference ◽  
Mrna Sequence ◽  
Thermodynamic Control ◽  
Base Pairs ◽  
Free Energy Difference ◽  
Ribosomal Frameshifting ◽  
Reading Frame ◽  
Slippery Sequence

Abstract mRNA contexts containing a ‘slippery’ sequence and a downstream secondary structure element stall the progression of the ribosome along the mRNA and induce its movement into the −1 reading frame. In this study we build a thermodynamic model based on Bayesian statistics to explain how −1 programmed ribosome frameshifting can work. As training sets for the model, we measured frameshifting efficiencies on 64 dnaX mRNA sequence variants in vitro and also used 21 published in vivo efficiencies. With the obtained free-energy difference between mRNA-tRNA base pairs in the 0 and −1 frames, the frameshifting efficiency of a given sequence can be reproduced and predicted from the tRNA−mRNA base pairing in the two frames. Our results further explain how modifications in the tRNA anticodon modulate frameshifting and show how the ribosome tunes the strength of the base-pair interactions.

scholarly journals A −1 Ribosomal Frameshift in the Transcript That Encodes the Major Head Protein of Bacteriophage A2 Mediates Biosynthesis of a Second Essential Component of the Capsid

2004 ◽  
Vol 186 (6) ◽  
pp. 1714-1719 ◽  
Author(s):  
Pilar García ◽  
Isabel Rodríguez ◽  
Juan E. Suárez
Keyword(s):  
Amino Acids ◽  
Mitomycin C ◽  
Proteolytic Processing ◽  
The Other ◽  
Capsid Proteins ◽  
Ribosomal Frameshift ◽  
Drastic Reduction ◽  
Stem Loop ◽  
Head Protein ◽  
Slippery Sequence

ABSTRACT The two major capsid proteins of Lactobacillus bacteriophage A2 share their amino termini. The smaller of these (gp5A) results from translation of orf5 and proteolytic processing after residue 123. The larger form (gp5B) originates through a −1 ribosomal frameshift at the penultimate codon of orf5 mRNA, resulting in a product that is 85 amino acids longer than gp5A. Frameshifting needs two cis-acting elements: a slippery region with the sequence C CCA AAA (0 frame), and a stem-loop that begins 9 nucleotides after the end of the slippery sequence. Mutations introduced in the slippery sequence suppress the frameshift. Similarly, deletion of the second half of the stem-loop results in drastic reduction of frameshifting. Both gp5A and gp5B appear to be essential for phage viability, since lysogens harboring prophages that produce only one or the other protein become lysed upon induction with mitomycin C, though no viable phage progeny are observed.

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