The Experts below are selected from a list of 471 Experts worldwide ranked by ideXlab platform
Samuel E. Butcher - One of the best experts on this subject based on the ideXlab platform.
-
HIV-1 frameshift efficiency is primarily determined by the stability of base pairs positioned at the mRNA entrance channel of the ribosome
Nucleic Acids Research, 2012Co-Authors: Kathryn D. Mouzakis, Andrew L. Lang, Kirk A. Vander Meulen, Preston D. Easterday, Samuel E. ButcherAbstract: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.
-
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...
-
Selection and characterization of small molecules that bind the HIV-1 frameshift site RNA.
ACS Chemical Biology, 2009Co-Authors: Ryan J. Marcheschi, Kathryn D. Mouzakis, Samuel E. ButcherAbstract:HIV-1 requires a −1 translational frameshift to properly synthesize the viral enzymes required for replication. The frameshift mechanism is dependent upon two RNA elements, a seven-nucleotide Slippery Sequence (UUUUUUA) and a downstream RNA structure. Frameshifting occurs with a frequency of ∼5%, and increasing or decreasing this frequency may result in a decrease in viral replication. Here, we report the results of a high-throughput screen designed to find small molecules that bind to the HIV-1 frameshift site RNA. Out of 34,500 compounds screened, 202 were identified as positive hits. We show that one of these compounds, doxorubicin, binds the HIV-1 RNA with low micromolar affinity (Kd = 2.8 μM). This binding was confirmed and localized to the RNA using NMR. Further analysis revealed that this compound increased the RNA stability by approximately 5 °C and decreased translational frameshifting by 28% (±14%), as measured in vitro.
-
Programmed Ribosomal Frameshifting in SIV Is Induced by a Highly Structured RNA Stem–Loop
Journal of Molecular Biology, 2007Co-Authors: Ryan J. Marcheschi, David W. Staple, Samuel E. ButcherAbstract:Abstract Simian immunodeficiency virus (SIV), like its human homologues (HIV-1, HIV-2), requires a -1 translational frameshift event to properly synthesize all of the proteins required for viral replication. The frameshift mechanism is dependent upon a seven-nucleotide Slippery Sequence and a downstream RNA structure. In SIV, the downstream RNA structure has been proposed to be either a stem–loop or a pseudoknot. Here, we report the functional, structural and thermodynamic characterization of the SIV frameshift site RNA. Translational frameshift assays indicate that a stem–loop structure is sufficient to promote efficient frameshifting in vitro. NMR and thermodynamic studies of SIV RNA constructs of varying length further support the absence of any pseudoknot interaction and indicate the presence of a stable stem–loop structure. We determined the structure of the SIV frameshift-inducing RNA by NMR. The structure reveals a highly ordered 12 nucleotide loop containing a sheared G-A pair, cross-strand adenine stacking, two G-C base-pairs, and a novel CCC triloop turn. The loop structure and its high thermostability preclude pseudoknot formation. Sequence conservation and modeling studies suggest that HIV-2 RNA forms the same structure. We conclude that, like the main sub-groups of HIV-1, SIV and HIV-2 utilize stable stem–loop structures to function as a thermodynamic barrier to translation, thereby inducing ribosomal pausing and frameshifting.
-
Solution structure and thermodynamic investigation of the HIV-1 frameshift inducing element.
Journal of Molecular Biology, 2005Co-Authors: David W. Staple, Samuel E. ButcherAbstract:Expression of the HIV reverse transcriptase and other essential viral enzymes requires a −1 translational frameshift. The frameshift event is induced by two highly conserved RNA elements within the HIV-1 mRNA: a UUUUUUA heptamer known as the Slippery Sequence, and a downstream RNA structure. Here, we report structural and thermodynamic evidence that the HIV-1 frameshift site RNA forms a stem-loop and lower helix separated by a three-purine bulge. We have determined the structure of the 45 nucleotide frameshift site RNA using multidimensional heteronuclear nuclear magnetic resonance (NMR) methods. The upper helix is highly thermostable (Tm>90 °C), forming 11 Watson–Crick base-pairs capped by a stable ACAA tetraloop. The eight base-pair lower helix was found to be only moderately stable (Tm=47 °C). A three-purine bulge separates the highly stable upper helix from the lower helix. Base stacking in the bulge forms a wedge, introducing a 60° bend between the helices. Interestingly, this bend is similar to those seen in a number of frameshift inducing pseudoknots for which structures have been solved. The lower helix must denature to allow the ribosome access to the Slippery site, but likely functions as a positioning element that enhances frameshift efficiency.
Ian Brierley - One of the best experts on this subject based on the ideXlab platform.
-
Programmed -2/-1 ribosomal frameshifting in simarteriviruses: An evolutionarily conserved mechanism
Journal of Virology, 2019Co-Authors: Yanhua Li, Ian Brierley, Sawsan Napthine, Andrew E. Firth, Tao Wang, Jens H. Kuhn, Ying FangAbstract:The −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 vitro translation 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. IMPORTANCE Simarteriviruses 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.
-
Characterization of the stimulators of protein-directed ribosomal frameshifting in Theiler's murine encephalomyelitis virus
Nucleic Acids Research, 2019Co-Authors: Sawsan Napthine, Ian Brierley, Susanne Bell, C.h. Hill, Andrew E. FirthAbstract:Many viruses utilize programmed –1 ribosomal frameshifting (–1 PRF) to express additional proteins or to produce frameshift and non-frameshift protein products at a fixed stoichiometric ratio. PRF is also utilized in the expression of a small number of cellular genes. Frameshifting is typically stimulated by signals contained within the mRNA: a ‘Slippery’ Sequence and a 3′-adjacent RNA structure. Recently, we showed that −1 PRF in encephalomyocarditis virus (EMCV) is trans-activated by the viral 2A protein, leading to a temporal change in PRF efficiency from 0% to 70% during virus infection. Here we analyzed PRF in the related Theiler's murine encephalomyelitis virus (TMEV). We show that 2A is also required for PRF in TMEV and can stimulate PRF to levels as high as 58% in rabbit reticulocyte cell-free translations and 81% during virus infection. We also show that TMEV 2A trans-activates PRF on the EMCV signal but not vice versa. We present an extensive mutational analysis of the frameshift stimulators (mRNA signals and 2A protein) analysing activity in in vitro translation, electrophoretic mobility shift and in vitro ribosome pausing assays. We also investigate the PRF mRNA signal with RNA structure probing. Our results substantially extend previous characterization of protein-stimulated PRF.
-
Spacer-length dependence of programmed −1 or −2 ribosomal frameshifting on a U6A heptamer supports a role for messenger RNA (mRNA) tension in frameshifting
Nucleic Acids Research, 2012Co-Authors: Robert J. C. Gilbert, Ian BrierleyAbstract:Programmed -1 ribosomal frameshifting is employed in the expression of a number of viral and cellular genes. In this process, the ribosome slips backwards by a single nucleotide and continues translation of an overlapping reading frame, generating a fusion protein. Frameshifting signals comprise a heptanucleotide Slippery Sequence, where the ribosome changes frame, and a stimulatory RNA structure, a stem-loop or RNA pseudoknot. Antisense oligonucleotides annealed appropriately 3' of a Slippery Sequence have also shown activity in frameshifting, at least in vitro. Here we examined frameshifting at the U6A Slippery Sequence of the HIV gag/pol signal and found high levels of both -1 and -2 frameshifting with stem-loop, pseudoknot or antisense oligonucleotide stimulators. By examining -1 and -2 frameshifting outcomes on mRNAs with varying Slippery Sequence-stimulatory RNA spacing distances, we found that -2 frameshifting was optimal at a spacer length 1-2 nucleotides shorter than that optimal for -1 frameshifting with all stimulatory RNAs tested. We propose that the shorter spacer increases the tension on the mRNA such that when the tRNA detaches, it more readily enters the -2 frame on the U6A heptamer. We propose that mRNA tension is central to frameshifting, whether promoted by stem-loop, pseudoknot or antisense oligonucleotide stimulator.
-
Pseudoknot-Dependent Programmed —1 Ribosomal Frameshifting: Structures, Mechanisms and Models
Recoding: Expansion of Decoding Rules Enriches Gene Expression, 2009Co-Authors: Ian Brierley, Robert J. C. Gilbert, Simon PennellAbstract:Programmed —1 ribosomal frameshifting is a translational recoding strategy that takes place during the elongation phase of protein biosynthesis. Frameshifting occurs in response to specific signals in the mRNA; a Slippery Sequence, where the ribosome changes frame, and a stimulatory RNA secondary structure, usually a pseudoknot, located immediately downstream. During the frameshift the ribosome slips backwards by a single nucleotide (in the 5′-wards/—1 direction) and continues translation in the new, overlapping reading frame, generating a fusion protein composed of the products of both the original and the —1 frame coding regions. In eukaryotes, frameshifting is largely a phenomenon of virus gene expression and associated predominantly with the expression of viral replicases. Research on frameshifting impacts upon diverse topics, including the ribosomal elongation cycle, RNA structure and function, tRNA modification, virus replication, antiviral intervention, evolution and bioinformatics. This chapter focuses on the structure and function of frameshift-stimulatory RNA pseudoknots and mechanistic aspects of ribosomal frameshifting. A variety of models of the frameshifting process are discussed in the light of recent advances in our understanding of ribosome structure and the elongation cycle.
-
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.
Kathryn Mouzakis - One of the best experts on this subject based on the ideXlab platform.
-
Minimal RNA Sequence Requirement of the HTLV-1 gag-pro RNA Frameshift Site
2016Co-Authors: Rebecca Salamon, Lee Jamie, Kathryn MouzakisAbstract:Human T-cell leukemia Virus Type I (HTLV-1) was the first identified human retrovirus, identified in 1980 (1). Infection with HTLV-1 results in adult T-cell leukemia with 5-10% incidence. An estimated 15-20 million individuals worldwide are infected with HTLV-1 (2). Replication of retroviruses, such as HTLV, is dependent upon synthesis of viral structural and enzymatic proteins. Synthesis of HTLV’s enzymatic proteins is dependent upon two -1 programmed ribosomal frameshift (PRF) events. PRF is defined as a programmed change in the ribosome’s reading frame during translation. During a -1 PRF, the ribosome shifts one nucleotide in the 5’ direction along the RNA when it is positioned on a ‘Slippery Sequence’. Slippery Sequences have a consensus Sequence of XXXYYYZ, where X can be any nucleotide, Y can be A or U, and Z cannot be G. This Sequence of nucleotides allows favorable tRNA anticodon:codon base-pairing between the tRNAs and mRNA in both the original (0) or alternate (-1) reading frames. In this work, the HTLV-1 gag-pro -1 frameshift site requirements are investigated. The gag-pro frameshift site consists of a heptanucleotide Slippery Sequence (AAAAAAC), followed by a downstream structure. A stem-loop structure is predicted downstream of the Slippery Sequence (3). We are investigating the minimal RNA Sequence required for frameshifting. To determine the minimal Sequence requirements, we designed two variant frameshift sites. The extended Sequence has an additional 126 nucleotides upstream and downstream of the frameshift site. In contrast, the minimal Sequence only includes 30 upstream and downstream of the frameshift site. We used cloning techniques to ligate a DNA insert coding for each frameshift site into the p2luc vector between the regions coding for two luciferase proteins - Firefly (fluc) and Renilla (rluc). In the p2luc vector, fluc is in the -1 reading frame relative to rluc. At this time, we have cloned the extended frameshift site into the p2luc vector. In order to Sequence the plasmid DNA, we amplified a 700 nucleotide section of the plasmid using a method called “Slowdown PCR”. This allowed the DNA polymerase to properly amplify the GC rich region within the frameshift site Sequence. This method, used in cis with a decreased denaturation temperature of 90 degrees Celsius for the sequencing reaction, allowed us to Sequence the 700 nucleotide PCR product. These results were compared to our intended plasmid Sequence and we were able to verify successful plasmid DNA cloning. In future experiments, we will amplify this DNA, use it to synthesize RNA, and then measure the in vitro frameshift efficiency for the extended frameshift site in triplicate. These experiments will be repeated for the minimal frameshift site discussed above. Frameshift efficiencies will be compared to published values for the HTLV-II gag-pro frameshift site. Based on the results, the effect of the upstream and downstream Sequence on frameshift efficiency can be determined.
-
Ribosomal Frameshifting in HTLV-1: Examining the pro-pol frameshift site
2016Co-Authors: Hector Caldera, Devon Chadeayne, Forrest Eagle, Evita Martin, Jeovanna Rios, Melanie Walker, Kathryn MouzakisAbstract:Human t-cell lymphotropic virus type l (HTLV-1) was the first identified human retrovirus, identified in 1980. Infection with HTLV-1 results in adult T-cell leukemia with 5-10% incidence. An estimated 15-20 million individuals worldwide are infected with HTLV. Replication of retroviruses, such as HTLV, is dependent upon synthesis of viral structural and enzymatic proteins. Synthesis of HTLV’s enzymatic proteins (Protease (PR), Reverse Transcriptase (RT), and Integrase (IN)) is dependent upon programmed ribosomal frameshifting (PRF). PRF is defined as a programmed change in the ribosome’s reading frame during translation. HTLV-1 has been observed to have ribosomal frameshifting at two difference sites. The frameshift sites gag-pro and pro-pol have been established but the efficiencies and structures of these two frameshift sites has not yet been determined. The HTLV-1 pro-pol site consists of three RNA elements: a Slippery Sequence (UUUAAAC), a spacer, and a downstream structure. In this work, the HTLV pro-pol -1PRF mechanism is investigated. A pseudoknot structure is predicted downstream of the Slippery Sequence. We hypothesize the pseudoknot structure contributes significantly to the frameshift efficiency. To test this hypothesis, we designed four variant frameshift sites to test the importance of the pseudoknot structure to frameshifting. An in vitro dual-luciferase frameshift assay was utilized to determine the frameshift efficiencies for the wild-type and variant frameshift sites.
-
Determination of the HTLV-1 pro-pol Frameshift Site Secondary Structure
The FASEB Journal, 2015Co-Authors: Kathryn Durnford, Erich G. Chapman, Antonia Atene, Amanda Broad, Jason M. Mackenzie, Dan Yeager, Jeffrey S. Kieft, Kathryn MouzakisAbstract:Human t-cell leukemia virus type 1 (HTLV-1) is a retrovirus that targets CD4+ T-cells in humans. Expression of human t-cell leukemia virus type I (HTLV-I) enzymes requires two -1 programmed ribosomal frameshifts (PRFs). These events occur between the gag-pro and pro-pol open reading frames. Each frameshift site includes a heptanucleotide Slippery Sequence followed by a downstream structure, which act in cis to produce specific frameshift efficiencies. While the -1 PRF and Slippery Sequences of these frameshift sites have been established in HTLV-I, the secondary structures have not been determined. In the pro-pol frameshift site, an RNA pseudoknot is predicted to fold downstream of the UUUAAAC Slippery Sequence. However, no structural data exists for this RNA. Here, we report a preliminary structure of the HTLV-1 pro-pol frameshift site RNA. Nucleotide reactivity data acquired from selective 2’-hydroxyl acylation experiments analyzed by primer extension (SHAPE) is consistent with a pseudoknot secondary structure. These results suggest the existence of a pseudoknot structure in the HTLV-1 pro-pol frameshift site.
-
Structure and Function of the HTLV-1 pro-pol Frameshift Site
The FASEB Journal, 2015Co-Authors: Devon Chadeayne, Summer Davis, Shawn Greyeyes, Allison Knewitz, Jordan Stelmaszek, Kathryn MouzakisAbstract:Human T-cell leukemia Virus Type I (HTLV-1) was the first identified human retrovirus, identified in 1980 (1). Infection with HTLV-1 results in adult T-cell leukemia with 5-10% incidence. An estimated 15-20 million individuals worldwide are infected with HTLV. Replication of retroviruses, such as HTLV, is dependent upon synthesis of viral structural and enzymatic proteins. Synthesis of HTLV’s enzymatic proteins (Protease (PR), Reverse Transcriptase (RT), and Integrase (IN)) is dependent upon programmed ribosomal frameshifting (PRF). PRF is defined by a programmed change in the ribosome’s reading frame during translation. In this work, HTLV-1 pro-pol -1 PRF is investigated. The pro-pol frameshift site consists of a heptanucleotide Slippery Sequence (UUUAAAC) followed by a downstream structure. The frameshift efficiency at this site is ~10% (2). A pseudoknot structure is predicted downstream of the Slippery Sequence (3). We hypothesize that the pseudoknot structure contributes significantly to the frameshift efficiency. To test this hypothesis, we designed four variant frameshift sites to test the importance of the pseudoknot structure to frameshifting. An in vitro dual-luciferase frameshift assay will be utilized to determine the frameshift efficiencies for the wild-type and variant frameshift sites. We report successful cloning of all of the plasmid DNAs, which code for the experimental and control RNAs used in the dual-luciferase frameshift assay. Eight of the ten plasmid DNAs has been successfully linearized and used for RNA synthesis and subsequently purified. Future work will include the synthesis and purification of the remaining RNAs, and final determination of the in vitro frameshift efficiency for each site.
-
The Effects of Local and Global RNA Stability on Frameshift Efficiency in the HTLV-II gag-pro Frameshift Site
2014Co-Authors: Melvina Lake, Serena Mancha, Kathryn MouzakisAbstract:Ribosomal frameshifting is defined as a shift in reading frame. During translation, a ribosomal frameshift (FS) can occur when the ribosome encounters a Slippery Sequence followed by a stable secondary structure. A Slippery Sequence specifically allows for the opportunity for tRNA repainring with the codons in the A- and P- sites of the ribosome. Retroviruses include programmed ribosomal frameshift (PRF) sites within their RNA, which increase viral genomic coding capacity by allowing translation of multiple reading frames from a single RNA. Human T-cell leukemia virus type-II (HTLV-II) uses PRF sites to translate its enzymatic proteins. These proteins are encoded in the pro and pol open reading frames. The first HTLV-II PRF site is located at the end of the gag open reading frame and is described as the gag-pro PRF site. This FS site contains a heptanucleotide Slippery Sequence (AAAAAAC) followed by a stem-loop structure. Recently, a study showed that local stability of the secondary structure was correlated to frameshift efficiency in HIV-1. We hypothesize that local stability has greater control of frameshift efficiency than global stability in the gap-pro PRF site in HTLV-II. To test this hypothesis, five variant stem-loops were designed to decouple local and global stability. There variant frameshift sites were inserted into a dual-luciferase reporter plasmid using molecular cloning techniques. Successful cloning was verified by DNA sequencing. These plasmid DNAs will be used for in vitro transcription of RNA. The resulting RNAs will be purified and utilized in an in vitro translation assay to determine the frameshift efficiencies for each variant frameshift site. Finally, the frameshift efficiencies will be compared to each other to determine if local or global stability has a stronger effect on frameshift efficiency.
Léa Brakier-gingras - One of the best experts on this subject based on the ideXlab platform.
-
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...
-
The three transfer RNAs occupying the A, P and E sites on the ribosome are involved in viral programmed -1 ribosomal frameshift
Nucleic Acids Research, 2007Co-Authors: Mélissa Léger, Dominic Dulude, Sergey V. Steinberg, Léa Brakier-gingrasAbstract:The -1 programmed ribosomal frameshifts (PRF), which are used by many viruses, occur at a heptanucleotide Slippery Sequence and are currently thought to involve the tRNAs interacting with the ribosomal P- and A-site codons. We investigated here whether the tRNA occupying the ribosomal E site that precedes a Slippery site influences -1 PRF. Using the human immunodeficiency virus type 1 (HIV-1) frameshift region, we found that mutating the E-site codon altered the -1 PRF efficiency. When the HIV-1 Slippery Sequence was replaced with other viral Slippery Sequences, mutating the E-site codon also altered the -1 PRF efficiency. Because HIV-1 -1 PRF can be recapitulated in bacteria, we used a bacterial ribosome system to select, by random mutagenesis, 16S ribosomal RNA (rRNA) mutations that modify the expression of a reporter requiring HIV-1 -1 PRF. Three mutants were isolated, which are located in helices 21 and 22 of 16S rRNA, a region involved in translocation and E-site tRNA binding. We propose a novel model where -1 PRF is triggered by an incomplete translocation and depends not only on the tRNAs interacting with the P- and A-site codons, but also on the tRNA occupying the E site.
-
Decreasing the frameshift efficiency translates into an equivalent reduction of the replication of the human immunodeficiency virus type 1
Virology, 2005Co-Authors: Dominic Dulude, Léa Brakier-gingras, Yamina A. Berchiche, Karine Gendron, Nikolaus HevekerAbstract:Abstract The Gag–Pol polyprotein of the human immunodeficiency virus type 1 (HIV-1) is the precursor of the virus enzymatic activities and is produced via a programmed −1 translational frameshift. In this study, we altered the frameshift efficiency by introducing mutations within the Slippery Sequence and the frameshift stimulatory signal, the two elements that control the frameshift. These mutations decreased the frameshift efficiency to different degrees, ranging from ∼0.3% to 70% of the wild-type efficiency. These values were mirrored by a reduced incorporation of Gag–Pol into virus-like particles, as assessed by a decrease in the reverse transcriptase activity associated to these particles. Analysis of Gag processing in infectious mutant virions revealed processing defects to various extents, with no clear correlation with frameshift decrease. Nevertheless, the observed frameshift reductions translated into equivalently reduced viral infectivity and replication kinetics. Our results show that even moderate variations in frameshift efficiency, as obtained with mutations in the frameshift stimulatory signal, reduce viral replication. Therapeutic targeting of this structure may therefore result in the attenuation of virus replication and in clinical benefit.
-
A reassessment of the response of the bacterial ribosome to the frameshift stimulatory signal of the human immunodeficiency virus type 1
RNA, 2004Co-Authors: Mélissa Léger, Sacha Sidani, Léa Brakier-gingrasAbstract:HIV-1 uses a programmed -1 ribosomal frameshift to produce the precursor of its enzymes. This frameshift occurs at a specific Slippery Sequence followed by a stimulatory signal, which was recently shown to be a two-stem helix, for which a three-purine bulge separates the upper and lower stems. In the present study, we investigated the response of the bacterial ribosome to this signal, using a translation system specialized for the expression of a firefly luciferase reporter. The HIV-1 frameshift region was inserted at the beginning of the coding Sequence of the luciferase gene, such that its expression requires a −1 frameshift. Mutations that disrupt the upper or the lower stem of the frameshift stimulatory signal or replace the purine bulge with pyrimidines decreased the frameshift efficiency, whereas compensatory mutations that re-form both stems restored the frame-shift efficiency to near wild-type level. These mutations had the same effect in a eukaryotic translation system, which shows that the bacterial ribosome responds like the eukaryote ribosome to the HIV-1 frameshift stimulatory signal. Also, we observed, in contrast to a previous report, that a stop codon immediately 3′ to the Slippery Sequence does not decrease the frameshift efficiency, ruling out a proposal that the frameshift involves the deacylated-tRNA and the peptidyl-tRNA in the E and P sites of the ribosome, rather than the peptidyl-tRNA and the aminoacyl-tRNA in the P and A sites, as commonly assumed. Finally, mutations in 16S ribosomal RNA that facilitate the accommodation of the incoming aminoacyl-tRNA in the A site decreased the frameshift efficiency, which supports a previous suggestion that the frameshift occurs when the aminoacyl-tRNA occupies the A/T entry site.
-
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.
Warren P Tate - One of the best experts on this subject based on the ideXlab platform.
-
the highly conserved codon following the Slippery Sequence supports 1 frameshift efficiency at the hiv 1 frameshift site
PLOS ONE, 2015Co-Authors: Suneeth F Mathew, Caillan Crowemcauliffe, Ryan Graves, Tony S Cardno, Cushla Mckinney, Elizabeth S Poole, Warren P TateAbstract:HIV-1 utilises −1 programmed ribosomal frameshifting to translate structural and enzymatic domains in a defined proportion required for replication. A Slippery Sequence, U UUU UUA, and a stem-loop are well-defined RNA features modulating −1 frameshifting in HIV-1. The GGG glycine codon immediately following the Slippery Sequence (the ‘intercodon’) contributes structurally to the start of the stem-loop but has no defined role in current models of the frameshift mechanism, as slippage is inferred to occur before the intercodon has reached the ribosomal decoding site. This GGG codon is highly conserved in natural isolates of HIV. When the natural intercodon was replaced with a stop codon two different decoding molecules—eRF1 protein or a cognate suppressor tRNA—were able to access and decode the intercodon prior to −1 frameshifting. This implies significant slippage occurs when the intercodon is in the (perhaps distorted) ribosomal A site. We accommodate the influence of the intercodon in a model of frame maintenance versus frameshifting in HIV-1.
-
Prokaryotic ribosomes recode the HIV-1 gag-pol -1 frameshift Sequence by an E/P site post-translocation simultaneous slippage mechanism
Nucleic Acids Research, 1995Co-Authors: Julie A. Horsfield, Daniel N. Wilson, Sally A. Mannering, Frances M. Adamski, Warren P TateAbstract:Abstract The mechanism favoured for -1 frameshifting at typical retroviral sites is a pre-translocation simultaneous slippage model. An alternative post-translocation mechanism would also generate the same protein Sequence across the frameshift site and therefore in this study the strategic placement of a stop codon has been used to distinguish between the two mechanisms. A 26 base pair frameshift Sequence from the HIV-1 gag-pol overlap has been modified to include a stop codon immediately 3' to the heptanucleotide frameshift signal, where it often occurs naturally in retroviral recoding sites. Stop codons at the 3'-end of the heptanucleotide Sequence decreased the frame-shifting efficiency on prokaryote ribosomes and the recording event was further depressed when the levels of the release factors in vivo were increased. In the presence of elevated levels of a defective release factor 2, frameshifting efficiency in vivo was increased in the constructs containing the stop codons recognized specifically by that release factor. These results are consistent with the last six nucleotides of the heptanucleotide Slippery Sequence occupying the ribosomal E and P sites, rather than the P and A sites, with the next codon occupying the A site and therefore with a post-translocation rather than a pre-translocation -1 slippage model.