The Experts below are selected from a list of 2778 Experts worldwide ranked by ideXlab platform
Jonathan D Dinman - One of the best experts on this subject based on the ideXlab platform.
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Programmed -1 Ribosomal Frameshifting in coronaviruses: A therapeutic target.
Virology, 2020Co-Authors: Jamie A Kelly, Michael T Woodside, Jonathan D DinmanAbstract:Human population growth, climate change, and globalization are accelerating the emergence of novel pathogenic viruses. In the past two decades alone, three such members of the coronavirus family have posed serious threats, spurring intense efforts to understand their biology as a way to identify targetable vulnerabilities. Coronaviruses use a programmed -1 Ribosomal Frameshift (-1 PRF) mechanism to direct synthesis of their replicase proteins. This is a critical switch in their replication program that can be therapeutically targeted. Here, we discuss how nearly half a century of research into -1 PRF have provided insight into the virological importance of -1 PRF, the molecular mechanisms that drive it, and approaches that can be used to manipulate it towards therapeutic outcomes with particular emphasis on SARS-CoV-2.
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structural and functional conservation of the programmed 1 Ribosomal Frameshift signal of sars coronavirus 2 sars cov 2
Journal of Biological Chemistry, 2020Co-Authors: Jamie A Kelly, Alexandra N Olson, Krishna Neupane, Sneha Munshi, Josue San Emeterio, Lois Pollack, Michael T Woodside, Jonathan D DinmanAbstract:Approximately 17 years after the severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic, the world is currently facing the COVID-19 pandemic caused by SARS corona virus 2 (SARS-CoV-2). According to the most optimistic projections, it will take more than a year to develop a vaccine, so the best short-term strategy may lie in identifying virus-specific targets for small molecule-based interventions. All coronaviruses utilize a molecular mechanism called programmed -1 Ribosomal Frameshift (-1 PRF) to control the relative expression of their proteins. Previous analyses of SARS-CoV have revealed that it employs a structurally unique three-stemmed mRNA pseudoknot that stimulates high -1 PRF rates and that it also harbors a -1 PRF attenuation element. Altering -1 PRF activity impairs virus replication, suggesting that this activity may be therapeutically targeted. Here, we comparatively analyzed the SARS-CoV and SARS-CoV-2 Frameshift signals. Structural and functional analyses revealed that both elements promote similar -1 PRF rates and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablate -1 PRF activity. We noted that the upstream attenuator hairpin activity is also functionally retained in both viruses, despite differences in the primary sequence in this region. Small-angle X-ray scattering analyses indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 have the same conformation. Finally, a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit -1 PRF was similarly effective against -1 PRF in SARS-CoV-2, suggesting that such Frameshift inhibitors may be promising lead compounds to combat the current COVID-19 pandemic.
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structural and functional conservation of the programmed 1 Ribosomal Frameshift signal of sars cov 2
bioRxiv, 2020Co-Authors: Jamie A Kelly, Jonathan D DinmanAbstract:17 years after the SARS-CoV epidemic, the world is facing the COVID-19 pandemic. COVID-19 is caused by a coronavirus named SARS-CoV-2. Given the most optimistic projections estimating that it will take over a year to develop a vaccine, the best short-term strategy may lie in identifying virus-specific targets for small molecule interventions. All coronaviruses utilize a molecular mechanism called -1 PRF to control the relative expression of their proteins. Prior analyses of SARS-CoV revealed that it employs a structurally unique three-stemmed mRNA pseudoknot to stimulate high rates of -1 PRF, and that it also harbors a -1 PRF attenuation element. Altering -1 PRF activity negatively impacts virus replication, suggesting that this molecular mechanism may be therapeutically targeted. Here we present a comparative analysis of the original SARS-CoV and SARS-CoV-2 Frameshift signals. Structural and functional analyses revealed that both elements promote similar rates of -1 PRF and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablated -1 PRF activity. The upstream attenuator hairpin activity has also been functionally retained. Small-angle x-ray scattering indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 had the same conformation. Finally, a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit -1 PRF was similarly effective against -1 PRF in SARS-CoV-2, suggesting that such Frameshift inhibitors may provide promising lead compounds to counter the current pandemic.
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Ribosomal Frameshifting in the CCR5 mRNA is regulated by miRNAs and the NMD pathway
Nature, 2014Co-Authors: Ashton T. Belew, Arturas Meskauskas, Sharmishtha Musalgaonkar, Vivek M. Advani, Sergey O. Sulima, Wojciech K. Kasprzak, Bruce A. Shapiro, Jonathan D DinmanAbstract:Programmed −1 Ribosomal Frameshift (−1 PRF) signals redirect translating ribosomes to slip back one base on messenger RNAs. Although well characterized in viruses, how these elements may regulate cellular gene expression is not understood. Here we describe a −1 PRF signal in the human mRNA encoding CCR5, the HIV-1 co-receptor. CCR5 mRNA-mediated −1 PRF is directed by an mRNA pseudoknot, and is stimulated by at least two microRNAs. Mapping the mRNA–miRNA interaction suggests that formation of a triplex RNA structure stimulates −1 PRF. A −1 PRF event on the CCR5 mRNA directs translating ribosomes to a premature termination codon, destabilizing it through the nonsense-mediated mRNA decay pathway. At least one additional mRNA decay pathway is also involved. Functional −1 PRF signals that seem to be regulated by miRNAs are also demonstrated in mRNAs encoding six other cytokine receptors, suggesting a novel mode through which immune responses may be fine-tuned in mammalian cells. Programmed −1 Ribosomal Frameshifting (−1 PRF) is a process by which a signal in a messenger RNA causes a translating ribosome to shift by one nucleotide, thus changing the reading frame; here −1 PRF in the mRNA for the co-receptor for HIV-1, CCR5, is stimulated by two microRNAs and leads to degradation of the transcript by nonsense-mediated decay and at least one other decay pathway. This paper reports the identification of an operational programmed minus one Ribosomal Frameshift (−1 PRF) signal that shifts elongating ribosomes into a premature termination codon in the human CCR5 mRNA, suggesting that it may destabilize the CCR5 mRNA through the nonsense-mediated mRNA decay pathway. PRF events, in which a signal in an mRNA causes a translating ribosome to shift by one nucleotide, thereby changing the reading frame, have been widely studied in viruses but less is known about how they act in mammalian cells. In the experiments described here, CCR5-mediated −1 PRF is enhanced by two microRNAs, one of which binds the −1 PRF signal directly and results in its structural reorganization. Other cytokine receptors also exhibit the potential for miRNA-regulated −1 PRF. These findings demonstrate a novel mechanism for fine-tuning immune responses in mammalian cells.
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endogenous Ribosomal Frameshift signals operate as mrna destabilizing elements through at least two molecular pathways in yeast
Nucleic Acids Research, 2011Co-Authors: Ashton T. Belew, Vivek M. Advani, Jonathan D DinmanAbstract:Although first discovered in viruses, previous studies have identified operational � 1 Ribosomal Frameshifting (� 1 RF) signals in eukaryotic genomic sequences, and suggested a role in mRNA stability. Here, four yeast � 1 RF signals are shown to promote significant mRNA destabilization through the nonsense mediated mRNA decay pathway (NMD), and genetic evidence is presented suggesting that they may also operate through the no-go decay pathway (NGD) as well. Yeast EST2 mRNA is highly unstable and contains up to five � 1 RF signals. Ablation of the � 1 RF signals or of NMD stabilizes this mRNA, and changes in � 1 RF efficiency have opposing effects on the steady-state abundance of the EST2 mRNA. These results demonstrate that endogenous � 1 RF signals function as mRNA destabilizing elements through at least two molecular pathways in yeast. Consistent with current evolutionary theory, phylogenetic analyses suggest that � 1 RF signals are rapidly evolving cis-acting regulatory elements. Identification of high confidence � 1 RF signals in � 10% of genes in all eukaryotic genomes surveyed suggests that � 1R F is a broadly used post-transcriptional regulator of gene expression.
Jamie A Kelly - One of the best experts on this subject based on the ideXlab platform.
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Programmed -1 Ribosomal Frameshifting in coronaviruses: A therapeutic target.
Virology, 2020Co-Authors: Jamie A Kelly, Michael T Woodside, Jonathan D DinmanAbstract:Human population growth, climate change, and globalization are accelerating the emergence of novel pathogenic viruses. In the past two decades alone, three such members of the coronavirus family have posed serious threats, spurring intense efforts to understand their biology as a way to identify targetable vulnerabilities. Coronaviruses use a programmed -1 Ribosomal Frameshift (-1 PRF) mechanism to direct synthesis of their replicase proteins. This is a critical switch in their replication program that can be therapeutically targeted. Here, we discuss how nearly half a century of research into -1 PRF have provided insight into the virological importance of -1 PRF, the molecular mechanisms that drive it, and approaches that can be used to manipulate it towards therapeutic outcomes with particular emphasis on SARS-CoV-2.
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structural and functional conservation of the programmed 1 Ribosomal Frameshift signal of sars coronavirus 2 sars cov 2
Journal of Biological Chemistry, 2020Co-Authors: Jamie A Kelly, Alexandra N Olson, Krishna Neupane, Sneha Munshi, Josue San Emeterio, Lois Pollack, Michael T Woodside, Jonathan D DinmanAbstract:Approximately 17 years after the severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic, the world is currently facing the COVID-19 pandemic caused by SARS corona virus 2 (SARS-CoV-2). According to the most optimistic projections, it will take more than a year to develop a vaccine, so the best short-term strategy may lie in identifying virus-specific targets for small molecule-based interventions. All coronaviruses utilize a molecular mechanism called programmed -1 Ribosomal Frameshift (-1 PRF) to control the relative expression of their proteins. Previous analyses of SARS-CoV have revealed that it employs a structurally unique three-stemmed mRNA pseudoknot that stimulates high -1 PRF rates and that it also harbors a -1 PRF attenuation element. Altering -1 PRF activity impairs virus replication, suggesting that this activity may be therapeutically targeted. Here, we comparatively analyzed the SARS-CoV and SARS-CoV-2 Frameshift signals. Structural and functional analyses revealed that both elements promote similar -1 PRF rates and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablate -1 PRF activity. We noted that the upstream attenuator hairpin activity is also functionally retained in both viruses, despite differences in the primary sequence in this region. Small-angle X-ray scattering analyses indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 have the same conformation. Finally, a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit -1 PRF was similarly effective against -1 PRF in SARS-CoV-2, suggesting that such Frameshift inhibitors may be promising lead compounds to combat the current COVID-19 pandemic.
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structural and functional conservation of the programmed 1 Ribosomal Frameshift signal of sars cov 2
bioRxiv, 2020Co-Authors: Jamie A Kelly, Jonathan D DinmanAbstract:17 years after the SARS-CoV epidemic, the world is facing the COVID-19 pandemic. COVID-19 is caused by a coronavirus named SARS-CoV-2. Given the most optimistic projections estimating that it will take over a year to develop a vaccine, the best short-term strategy may lie in identifying virus-specific targets for small molecule interventions. All coronaviruses utilize a molecular mechanism called -1 PRF to control the relative expression of their proteins. Prior analyses of SARS-CoV revealed that it employs a structurally unique three-stemmed mRNA pseudoknot to stimulate high rates of -1 PRF, and that it also harbors a -1 PRF attenuation element. Altering -1 PRF activity negatively impacts virus replication, suggesting that this molecular mechanism may be therapeutically targeted. Here we present a comparative analysis of the original SARS-CoV and SARS-CoV-2 Frameshift signals. Structural and functional analyses revealed that both elements promote similar rates of -1 PRF and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablated -1 PRF activity. The upstream attenuator hairpin activity has also been functionally retained. Small-angle x-ray scattering indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 had the same conformation. Finally, a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit -1 PRF was similarly effective against -1 PRF in SARS-CoV-2, suggesting that such Frameshift inhibitors may provide promising lead compounds to counter the current pandemic.
Ian Brierley - One of the best experts on this subject based on the ideXlab platform.
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Structure-function analysis of the Ribosomal Frameshifting signal of two human immunodeficiency virus type 1 isolates with increased resistance to viral protease inhibitors.
Journal of General Virology, 2007Co-Authors: Roseanne Girnary, Louise King, Laurence H Robinson, Robert Elston, Ian BrierleyAbstract: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.
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Characterization of the Frameshift signal of Edr, a mammalian example of programmed −1 Ribosomal Frameshifting
Nucleic Acids Research, 2005Co-Authors: Emily Manktelow, Kenji Shigemoto, Ian BrierleyAbstract:The Ribosomal Frameshifting signal of the mouse embryonal carcinoma differentiation regulated (Edr) gene represents the sole documented example of programmed −1 Frameshifting in mammalian cellular genes [Shigemoto,K., Brennan,J., Walls,E,. Watson,C.J., Stott,D., Rigby,P.W. and Reith,A.D. (2001), Nucleic Acids Res., 29, 4079–4088]. Here, we have employed site-directed mutagenesis and RNA structure probing to characterize the Edr signal. We began by confirming the functionality and magnitude of the signal and the role of a GGGAAAC motif as the slippery sequence. Subsequently, we derived a model of the Edr stimulatory RNA and assessed its similarity to those stimulatory RNAs found at viral Frameshift sites. We found that the structure is an RNA pseudoknot possessing features typical of retroviral Frameshifter pseudoknots. From these experiments, we conclude that the Edr signal and by inference, the human orthologue PEG10, do not represent a novel ‘cellular class’ of programmed −1 Ribosomal Frameshift signal, but rather are similar to viral examples, albeit with some interesting features. The similarity to viral Frameshift signals may complicate the design of antiviral therapies that target the Frameshift process.
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Characterization of the Frameshift signal of Edr, a mammalian example of programmed −1 Ribosomal Frameshifting
Nucleic Acids Research, 2005Co-Authors: Emily Manktelow, Kazuhiro Shigemoto, Ian BrierleyAbstract:The Ribosomal Frameshifting signal of the mouse embryonal carcinoma differentiation regulated (Edr) gene represents the sole documented example of programmed −1 Frameshifting in mammalian cellular genes [Shigemoto,K., Brennan,J., Walls,E,. Watson,C.J., Stott,D., Rigby,P.W. and Reith,A.D. (2001), Nucleic Acids Res., 29, 4079–4088]. Here, we have employed site-directed mutagenesis and RNA structure probing to characterize the Edr signal. We began by confirming the functionality and magnitude of the signal and the role of a GGGAAAC motif as the slippery sequence. Subsequently, we derived a model of the Edr stimulatory RNA and assessed its similarity to those stimulatory RNAs found at viral Frameshift sites. We found that the structure is an RNA pseudoknot possessing features typical of retroviral Frameshifter pseudoknots. From these experiments, we conclude that the Edr signal and by inference, the human orthologue PEG10, do not represent a novel ‘cellular class’ of programmed −1 Ribosomal Frameshift signal, but rather are similar to viral examples, albeit with some interesting features. The similarity to viral Frameshift signals may complicate the design of antiviral therapies that target the Frameshift process.
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secondary structure and mutational analysis of the Ribosomal Frameshift signal of rous sarcoma virus
Journal of Molecular Biology, 1998Co-Authors: Beate Marczinke, Alison J Bloys, Rosamond Fisher, Marijana Vidakovic, Ian BrierleyAbstract:Abstract Expression of the Gag-Pol polyprotein of Rous sarcoma virus (RSV) requires a −1 Ribosomal Frameshifting event at the overlap region of the gag and pol open reading frames. The signal for Frameshifting is composed of two essential mRNA elements; a slippery sequence (AAAUUUA) where the ribosome changes reading frame, and a stimulatory RNA structure located immediately downstream. This RNA is predicted to be a complex stem-loop but may also form an RNA pseudoknot. We have investigated the structure of the RSV Frameshift signal by a combination of enzymatic and chemical structure probing and site-directed mutagenesis. The stimulatory RNA is indeed a complex stem-loop with a long stable stem and two additional stem-loops contained as substructures within the main loop region. The substructures are not however required for Frameshifting. Evidence for an additional interaction between a stretch of nucleotides in the main loop and a region downstream to generate an RNA pseudoknot was obtained from an analysis of the Frameshifting properties of RSV mutants translated in the rabbit reticulocyte lysate in vitro translation system. Mutations that disrupted the predicted pseudoknot-forming sequences reduced Frameshifting but when the mutations were combined and should re-form the pseudoknot, Frameshifting was restored to a level approaching that of the wild-type construct. It was also observed that the predicted pseudoknot-forming regions had reduced sensitivity to cleavage by the single-stranded probe imidazole. Overall, however, the structure probing data indicate that the pseudoknot interaction is weak and may form transiently. In comparison to other characterised RNA structures present at viral Frameshift signals, the RSV stimulator falls into a novel group. It cannot be considered to be a simple hairpin-loop yet it is distinct from other well characterised Frameshift-inducing RNA pseudoknots in that the overall contribution of the RSV pseudoknot to Frameshifting is less dramatic.
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Expression of a coronavirus Ribosomal Frameshift signal in Escherichia coli: influence of tRNA anticodon modification on Frameshifting
Journal of Molecular Biology, 1997Co-Authors: Ian Brierley, Michayla R Meredith, Alison J Bloys, Tord G HagervallAbstract:Abstract Eukaryotic Ribosomal Frameshift signals generally contain two elements, a heptanucleotide slippery sequence (XXXYYYN) and an RNA secondary structure, often an RNA pseudoknot, located downstream. Frameshifting takes place at the slippery sequence by simultaneous slippage of two ribosome-bound tRNAs. All of the tRNAs that are predicted to decode Frameshift sites in the Ribosomal A-site (XXXY YYN ) possess a hypermodified base in the anticodon-loop and it is conceivable that these modifications play a role in the Frameshift process. To test this, we expressed slippery sequence variants of the coronavirus IBV Frameshift signal in strains of Escherichia coli unable to modify fully either tRNALys or tRNAAsn. At the slippery sequences UUUA AAC and UUUA AAU (underlined codon decoded by tRNAAsn, anticodon 5′ QUU 3′), Frameshifting was very inefficient (2 to 3%) and in strains deficient in the biosynthesis of Q base, was increased (AAU) or decreased (AAC) only two-fold. In E. coli, therefore, hypomodification of tRNAAsn had little effect on Frameshifting. The situation with the efficient slippery sequences UUUA AAA (15%) and UUUA AAG (40%) (underlined codon decoded by tRNALys, anticodon 5′ mnm5s2UUU 3′) was more complex, since the wobble base of tRNALys is modified at two positions. Of four available mutants, only trmE (s2UUU) had a marked influence on Frameshifting, increasing the efficiency of the process at the slippery sequence UUUA AAA . No effect on Frameshifting was seen in trmC1 (cmnm5s2UUU) or trmC2 (nm5s2UUU) strains and only a very small reduction (at UUUA AAG ) was observed in an asuE (mnm5UUU) strain. The slipperiness of tRNALys, therefore, cannot be ascribed to a single modification site on the base. However, the data support a role for the amino group of the mnm5 substitution in shaping the anticodon structure. Whether these conclusions can be extended to eukaryotic translation systems is uncertain. Although E. coli ribosomes changed frame at the IBV signal (UUUAAAG) with an efficiency similar to that measured in reticulocyte lysates (40%), there were important qualitative differences. Frameshifting of prokaryotic ribosomes was pseudoknot-independent (although secondary structure dependent) and appeared to require slippage of only a single tRNA.
Léa Brakier-gingras - One of the best experts on this subject based on the ideXlab platform.
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Targeting Frameshifting in the human immunodeficiency virus
Expert Opinion on Therapeutic Targets, 2012Co-Authors: Léa Brakier-gingras, Johanie Charbonneau, Samuel E ButcherAbstract:Introduction: HIV-1 uses a programmed –1 Ribosomal Frameshift to generate Gag-Pol, the precursor of its enzymes, when its full-length mRNA is translated by the ribosomes of the infected cells. This change in the reading frame occurs at a so-called slippery sequence that is followed by a specific secondary structure, the Frameshift stimulatory signal. This signal controls the Frameshift efficiency. The synthesis of HIV-1 enzymes is critical for virus replication and therefore, the –1 Ribosomal Frameshift could be the target of novel antiviral drugs. Areas covered: Various approaches were used to select drugs interfering with the –1 Frameshift of HIV-1. These include the selection and modification of chemical compounds that specifically bind to the Frameshift stimulatory signal, the use of antisense oligonucleotides targeting this signal and the selection of compounds that modulate HIV-1 Frameshift, by using bicistronic reporters where the expression of the second cistron depends upon HIV-1 Frameshift. Expe...
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Programmed —1 Ribosomal Frameshift in the Human Immunodeficiency Virus of Type 1
Recoding: Expansion of Decoding Rules Enriches Gene Expression, 2009Co-Authors: Léa Brakier-gingras, Dominic DuludeAbstract:A programmed —1 Ribosomal Frameshift enables the human immunode-ficiency virus of type 1 (HIV-1) to produce its enzymes in a precise proportion relative to its structural proteins, which is necessary to control viral assembly and maturation. Here, we critically review models that account for this phenomenon, focusing on the most recent model, which postulates that the Frameshift is triggered by an incomplete translocation and involves the slippage of three tRNAs. The effect of changes in the rate of translation initiation and elongation and the possible involvement of cellular factors in Frameshifting are briefly examined. Finally, we highlight recent efforts intended to interfere with this type of Frameshift as a strategy to develop novel anti-HIV drugs.
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The Frameshift Stimulatory Signal of Human Immunodeficiency Virus Type 1 Group O is a Pseudoknot
Journal of Molecular Biology, 2003Co-Authors: Martin Baril, Dominic Dulude, Sergey V. Steinberg, Léa Brakier-gingrasAbstract:Abstract Human immunodeficiency virus type 1 (HIV-1) requires a programmed −1 Ribosomal Frameshift to produce Gag–Pol, the precursor of its enzymatic activities. This Frameshift occurs at a slippery sequence on the viral messenger RNA and is stimulated by a specific structure, downstream of the shift site. While in group M, the most abundant HIV-1 group, the Frameshift stimulatory signal is an extended bulged stem-loop, we show here, using a combination of mutagenesis and probing studies, that it is a pseudoknot in group O. The mutagenesis and probing studies coupled to an in silico analysis show that group O pseudoknot is a hairpin-type pseudoknot with two coaxially stacked stems of eight base-pairs (stem 1 and stem 2), connected by single-stranded loops of 2 nt (loop 1) and 20 nt (loop 2). Mutations impairing formation of stem 1 or stem 2 of the pseudoknot reduce Frameshift efficiency, whereas compensatory changes that allow re-formation of these stems restore the Frameshift efficiency to near wild-type level. The difference between the Frameshift stimulatory signal of group O and group M supports the hypothesis that these groups originate from a different monkey to human transmission.
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Characterization of the Frameshift stimulatory signal controlling a programmed –1 Ribosomal Frameshift in the human immunodeficiency virus type 1
Nucleic Acids Research, 2002Co-Authors: Dominic Dulude, Martin Baril, Léa Brakier-gingrasAbstract:Synthesis of the Gag-Pol protein of the human immunodeficiency virus type 1 (HIV-1) requires a programmed –1 Ribosomal Frameshifting when ribosomes translate the unspliced viral messenger RNA. This Frameshift occurs at a slippery sequence followed by an RNA structure motif that stimulates Frameshifting. This motif is commonly assumed to be a simple stem–loop for HIV-1. In this study, we show that the Frameshift stimulatory signal is more complex than believed and consists of a two-stem helix. The upper stem–loop corresponds to the classic stem–loop, and the lower stem is formed by pairing the spacer region following the slippery sequence and preceding this classic stem–loop with a segment downstream of this stem–loop. A three-purine bulge interrupts the two stems. This structure was suggested by enzymatic probing with nuclease V1 of an RNA fragment corresponding to the gag/pol Frameshift region of HIV-1. The involvement of the novel lower stem in Frameshifting was supported by site-directed mutagenesis. A fragment encompassing the gag/pol Frameshift region of HIV-1 was inserted in the beginning of the coding sequence of a reporter gene coding for the firefly luciferase, such that expression of luciferase requires a –1 Frameshift. When the reporter was expressed in COS cells, mutations that disrupt the capacity to form the lower stem reduced Frameshifting, whereas compensatory changes that allow re-formation of this stem restored the Frameshift efficiency near wild-type level. The two-stem structure that we propose for the Frameshift stimulatory signal of HIV-1 differs from the RNA triple helix structure recently proposed.
Dominic Dulude - One of the best experts on this subject based on the ideXlab platform.
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Programmed —1 Ribosomal Frameshift in the Human Immunodeficiency Virus of Type 1
Recoding: Expansion of Decoding Rules Enriches Gene Expression, 2009Co-Authors: Léa Brakier-gingras, Dominic DuludeAbstract:A programmed —1 Ribosomal Frameshift enables the human immunode-ficiency virus of type 1 (HIV-1) to produce its enzymes in a precise proportion relative to its structural proteins, which is necessary to control viral assembly and maturation. Here, we critically review models that account for this phenomenon, focusing on the most recent model, which postulates that the Frameshift is triggered by an incomplete translocation and involves the slippage of three tRNAs. The effect of changes in the rate of translation initiation and elongation and the possible involvement of cellular factors in Frameshifting are briefly examined. Finally, we highlight recent efforts intended to interfere with this type of Frameshift as a strategy to develop novel anti-HIV drugs.
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selection of peptides interfering with a Ribosomal Frameshift in the human immunodeficiency virus type 1
RNA, 2008Co-Authors: Dominic Dulude, Gabriel Thebergejulien, Lea Brakiergingras, Nikolaus HevekerAbstract:The human immunodeficiency virus of type 1 (HIV-1) uses a programmed -1 Ribosomal Frameshift to produce the precursor of its enzymes, and changes in Frameshift efficiency reduce replicative fitness of the virus. We used a fluorescent two-reporter system to screen for peptides that reduce HIV-1 Frameshift in bacteria, knowing that the Frameshift can be reproduced in Escherichia coli. Expression of one reporter, the green fluorescent protein (GFP), requires the HIV-1 Frameshift, whereas the second reporter, the red fluorescent protein (RFP), is used to assess normal translation. A peptide library biased for RNA binding was inserted into the sequence of the protein thioredoxin and expressed in reporter-containing bacteria, which were then screened by fluorescence-activated cell sorting (FACS). We identified peptide sequences that reduce Frameshift efficiency by over 50% without altering normal translation. The identified sequences are also active against different Frameshift stimulatory signals, suggesting that they bind a target important for Frameshifting in general, probably the ribosome. Successful transfer of active sequences to a different scaffold in a eukaryotic test system demonstrates that the anti-Frameshift activity of the peptides is neither due to scaffold-dependent conformation nor effects of the scaffold protein itself on Frameshifting. The method we describe identifies peptides that will provide useful tools to further study the mechanism of Frameshift and may permit the development of lead compounds of therapeutic interest.
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the presence of the tar rna structure alters the programmed 1 Ribosomal Frameshift efficiency of the human immunodeficiency virus type 1 hiv 1 by modifying the rate of translation initiation
Nucleic Acids Research, 2008Co-Authors: Karine Gendron, Dominic Dulude, Johanie Charbonneau, Nikolaus Heveker, Gerardo Ferbeyre, Lea BrakiergingrasAbstract:HIV-1 uses a programmed -1 Ribosomal Frameshift to synthesize the precursor of its enzymes, Gag-Pol. The Frameshift efficiency that is critical for the virus replication, is controlled by an interaction between the ribosome and a specific structure on the viral mRNA, the Frameshift stimulatory signal. The rate of cap-dependent translation initiation is known to be altered by the TAR RNA structure, present at the 5' and 3' end of all HIV-1 mRNAs. Depending upon its concentration, TAR activates or inhibits the double-stranded RNA-dependent protein kinase (PKR). We investigated here whether changes in translation initiation caused by TAR affect HIV-1 Frameshift efficiency. CD4+ T cells and 293T cells were transfected with a dual-luciferase construct where the firefly luciferase expression depends upon the HIV-1 Frameshift. Translation initiation was altered by adding TAR in cis or trans of the reporter mRNA. We show that HIV-1 Frameshift efficiency correlates negatively with changes in the rate of translation initiation caused by TAR and mediated by PKR. A model is presented where changes in the rate of initiation affect the probability of Frameshifting by altering the distance between elongating ribosomes on the mRNA, which influences the frequency of encounter between these ribosomes and the Frameshift stimulatory signal.
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efficiency of a programmed 1 Ribosomal Frameshift in the different subtypes of the human immunodeficiency virus type 1 group m
RNA, 2003Co-Authors: Martin Baril, Dominic Dulude, Karine Gendron, Guy Lemay, Lea BrakiergingrasAbstract:The synthesis of the Gag-Pol polyprotein, the precursor of the enzymes of the human immunodeficiency virus type 1 (HIV-1), requires a programmed −1 Ribosomal Frameshift. This Frameshift has been investigated so far only for subtype B of HIV-1 group M. In this subtype, the Frameshift stimulatory signal was found to be a two-stem helix, in which a three-purine bulge interrupts the two stems. In this study, using a luciferase reporter system, we compare, for the first time, the Frameshift efficiency of all the subtypes of group M. Mutants of subtype B, including a natural variant were also investigated. Our results with mutants of subtype B confirm that the bulge and the lower stem of the Frameshift stimulatory signal contribute to the Frameshift in addition to the upper stem–loop considered previously as the sole participant. Our results also show that the Frameshift stimulatory signal of all of the other subtypes of group M can be folded into the same structure as in subtype B, despite sequence variations. Moreover, the Frameshift efficiency of these subtypes, when assessed in cultured cells, falls within a narrow window (the maximal deviation from the mean value calculated from the experimental values of all the subtypes being ~35%), although the predicted thermodynamic stability of the Frameshift stimulatory signal differs between the subtypes (from −17.2 kcal/mole to −26.2 kcal/mole). The fact that the Frameshift efficiencies fall within a narrow range for all of the subtypes of HIV-1 group M stresses the potential of the Frameshift event as an antiviral target.
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The Frameshift Stimulatory Signal of Human Immunodeficiency Virus Type 1 Group O is a Pseudoknot
Journal of Molecular Biology, 2003Co-Authors: Martin Baril, Dominic Dulude, Sergey V. Steinberg, Léa Brakier-gingrasAbstract:Abstract Human immunodeficiency virus type 1 (HIV-1) requires a programmed −1 Ribosomal Frameshift to produce Gag–Pol, the precursor of its enzymatic activities. This Frameshift occurs at a slippery sequence on the viral messenger RNA and is stimulated by a specific structure, downstream of the shift site. While in group M, the most abundant HIV-1 group, the Frameshift stimulatory signal is an extended bulged stem-loop, we show here, using a combination of mutagenesis and probing studies, that it is a pseudoknot in group O. The mutagenesis and probing studies coupled to an in silico analysis show that group O pseudoknot is a hairpin-type pseudoknot with two coaxially stacked stems of eight base-pairs (stem 1 and stem 2), connected by single-stranded loops of 2 nt (loop 1) and 20 nt (loop 2). Mutations impairing formation of stem 1 or stem 2 of the pseudoknot reduce Frameshift efficiency, whereas compensatory changes that allow re-formation of these stems restore the Frameshift efficiency to near wild-type level. The difference between the Frameshift stimulatory signal of group O and group M supports the hypothesis that these groups originate from a different monkey to human transmission.
