Frameshift Efficiency Research Articles

HIV-1 frameshift efficiency is primarily determined by the stability of base pairs positioned at the mRNA entrance channel of the ribosome

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.

Programmed −1 frameshifting efficiency correlates with RNA pseudoknot conformational plasticity, not resistance to mechanical unfolding

Programmed -1 frameshifting, whereby the reading frame of a ribosome on messenger RNA is shifted in order to generate an alternate gene product, is often triggered by a pseudoknot structure in the mRNA in combination with an upstream slippery sequence. The efficiency of frameshifting varies widely for different sites, but the factors that determine frameshifting efficiency are not yet fully understood. Previous work has suggested that frameshifting efficiency is related to the resistance of the pseudoknot against mechanical unfolding. We tested this hypothesis by studying the mechanical properties of a panel of pseudoknots with frameshifting efficiencies ranging from 2% to 30%: four pseudoknots from retroviruses, two from luteoviruses, one from a coronavirus, and a nonframeshifting bacteriophage pseudoknot. Using optical tweezers to apply tension across the RNA, we measured the distribution of forces required to unfold each pseudoknot. We found that neither the average unfolding force, nor the unfolding kinetics, nor the parameters describing the energy landscape for mechanical unfolding of the pseudoknot (energy barrier height and distance to the transition state) could be correlated to frameshifting efficiency. These results indicate that the resistance of pseudoknots to mechanical unfolding is not a primary determinant of frameshifting efficiency. However, increased frameshifting efficiency was correlated with an increased tendency to form alternate, incompletely folded structures, suggesting a more complex picture of the role of the pseudoknot involving the conformational dynamics.

Net −1 frameshifting on a noncanonical sequence in a herpes simplex virus drug-resistant mutant is stimulated by nonstop mRNA

Ribosomal frameshifting entails slippage of the translational machinery during elongation. Frameshifting permits expression of more than one polypeptide from an otherwise monocistronic mRNA, and can restore expression of polypeptides in the face of frameshift mutations. A common mutation conferring acyclovir resistance in patients with herpes simplex virus disease deletes one cytosine from a run of six cytosines (C-chord) in the viral thymidine kinase (tk) gene. However, this mutation does not abolish TK activity, which is important for pathogenicity. To investigate how this mutant retains TK activity, we engineered and analyzed viruses expressing epitope-tagged TK. We found that the mutant's TK activity can be accounted for by low levels of full-length TK polypeptide produced by net -1 frameshifting during translation. The efficiency of frameshifting was relatively high, 3-5%, as the polypeptide from the reading frame generated by the deletion, which lacks stop codons (nonstop), was poorly expressed mainly because of inefficient protein synthesis. Stop codons introduced into this reading frame greatly increased its expression, but greatly decreased the level of full-length TK, indicating that frameshifting is strongly stimulated by a new mechanism, nonstop mRNA, which we hypothesize involves stalling of ribosomes on the polyA tail. Mutational studies indicated that frameshifting occurs on or near the C-chord, a region lacking a canonical slippery sequence. Nonstop stimulation of frameshifting also occurred when the C-chord was replaced with a canonical slippery sequence from HIV. This mechanism thus permits biologically and clinically relevant TK synthesis, and may occur more generally.

The 5′ UTR of HIV-1 full-length mRNA and the Tat viral protein modulate the programmed −1 ribosomal frameshift that generates HIV-1 enzymes

Translation of the full-length messenger RNA (mRNA) of the human immunodeficiency virus type 1 (HIV-1) generates the precursor of the viral enzymes via a programmed -1 ribosomal frameshift. Here, using dual-luciferase reporters, we investigated whether the highly structured 5' untranslated region (UTR) of this mRNA, which interferes with translation initiation, can modulate HIV-1 frameshift efficiency. We showed that, when the 5' UTR of HIV-1 mRNA occupies the 5' end of the reporter mRNA, HIV-1 frameshift efficiency is increased about fourfold in Jurkat T-cells, compared with a control dual-luciferase reporter with a short unstructured 5' UTR. This increase was related to an interference with cap-dependent translation initiation by the TAR-Poly(A) region at the 5' end of the messenger. HIV-1 mRNA 5' UTR also contains an internal ribosome entry site (IRES), but we showed that, when the cap-dependent initiation mode is available, the IRES is not used or is weakly used. However, when the ribosomes have to use the IRES to translate the dual-luciferase reporter, the frameshift efficiency is comparable to that of the control dual-luciferase reporter. The decrease in cap-dependent initiation and the accompanying increase in frameshift efficiency caused by the 5' UTR of HIV-1 mRNA is antagonized, in a dose-dependent way, by the Tat viral protein. Tat also stimulates the IRES-dependent initiation and decreases the corresponding frameshift efficiency. A model is presented that accounts for the variations in frameshift efficiency depending on the 5' UTR and the presence of Tat, and it is proposed that a range of frameshift efficiencies is compatible with the virus replication.

Frameshifting Efficiency is not Determined by the Mechanical Stability of RNA Pseudoknots

Ribosomes translate messenger RNA (mRNA) in 3-nucleotide steps, maintaining an open reading frame until a stop codon is reached. Programmed −1 translational frameshifting, whereby the ribosome is forced backward by 1 nt to shift the reading frame, occurs in many viruses. The resulting expression of two proteins from a single mRNA at a fixed ratio is essential for viral propagation. Frameshifting depends on two mRNA structures: a slippery sequence and a downstream pseudoknot. Recent work suggests frameshifting pseudoknots provide greater barriers to unfolding by the ribosomal helicase (1), likely a function of their increased mechanical stability. The mechanical stability of a panel of 8 pseudoknots, associated with different frameshifting efficiencies, was investigated by repeatedly unfolding and refolding single pseudoknot molecules held under tension by optical tweezers. Pseudoknot unfolding was characterized by measuring the associated molecular contour length changes, unfolding forces, and force-dependent kinetics. Most of the pseudoknots we examined unfold at high force (∼30-50 pN). The corresponding contour length changes were consistent with native pseudoknot unfolding. Most often the unfolding occurred in a single step, but for some pseudoknots the dominant unfolding pathway occurred via a partially-folded intermediate at lower force (∼15-20 pN). Surprisingly, we found no obvious correlation between frameshifting efficiency and pseudoknot mechanical stability, indicating that some other property must determine the frameshifting efficiency. These results extend our understanding of the mechanics of RNA pseudoknot structure formation, and the relation between pseudoknot folding and programmed translational frameshifting. (1) Namy, O., Moran, S.J., Stuart, D.I., Gilbert, R.J. & Brierley, I. A mechanical explanation of RNA pseudoknot function in programmed ribosomal frameshifting. Nature 441, 244-247 (2006).

Mutational patterns in the frameshift-regulating site of HIV-1 selected by protease inhibitors

Sustained suppression of viral replication in HIV-1 infected patients is especially hampered by the emergence of HIV-1 drug resistance. The mechanisms of drug resistance mainly involve mutations directly altering the interaction of viral enzymes and inhibitors. However, protease inhibitors do not only select for mutations in the protease but also for mutations in the precursor Gag and Pol proteins. In this study, we analysed the frameshift-regulating site of HIV-1 subtype B isolates, which also encodes for Gag and Pol proteins, classified as either treatment-naïve (TN) or protease inhibitor resistant (PI-R). HIV-1 Gag cleavage site mutations (G435E, K436N, I437V, L449F/V) especially correlated with protease inhibitor resistance mutations, but also Pol cleavage site mutations (D05G, D05S) could be assigned to specific protease resistance profiles. Additionally, two Gag non-cleavage site mutations (S440F, H441P) were observed more often in HIV-1 isolates carrying protease resistance mutations. However, in dual luciferase assays, the frameshift efficiencies of specific clones did not reveal any effect from these mutations. Nevertheless, two patterns of mutations modestly increased the frameshift rates in vitro, but were not specifically accumulating in PI-resistant HIV-1 isolates. In summary, HIV-1 Gag cleavage site mutations were dominantly selected in PI-resistant HIV-1 isolates but also Pol cleavage site mutations influenced resistance profiles in the protease. Additionally, Gag non-cleavage site mutations accumulated in PI-resistant HIV-1 isolates, but were not related to an increased frameshift efficiency.

Glucose signalling pathway controls the programmed ribosomal frameshift efficiency in retroviral‐like element Ty3 in Saccharomyces cerevisiae

Ty3 elements of S. cerevisiae contain two overlapping coding regions, GAG3 and POL3, which are functional homologues of retroviral gag and pol genes, respectively. Pol3 is translated as a Gag3‐Pol3 fusion protein dependent on a +1 programmed frameshift at a site with the overlap between the two genes. We show that the Ty3 frameshift frequency varies up to 10‐fold in S. cerevisiae cells depending on carbon source. Frameshift efficiency is significantly lower in cells growing on glucose as carbon source than in cells growing on poor alternative carbon sources (glycerol/lactate or galactose). Our results indicate that Ty3 programmed ribosomal frameshift efficiency in response to glucose signalling requires two protein kinases: Snf1p and cAMP‐dependent protein kinase A (PKA). Increased frameshifting on alternative carbon sources also appears to require cytoplasmic localization of Snf1p, mediated by the Sip2p protein. In addition to the two required protein kinases, our results implicate that Stm1p, a ribosome‐associated protein involved in nutrient sensing, is essential for the carbon source‐dependent regulation of Ty3 frameshifting. These data indicate that Ty3 programmed ribosomal frameshift is not a constitutive process but that it is regulated in response to the glucose‐signalling pathway. Copyright © 2011 John Wiley & Sons, Ltd.

Annexin A2 Binds RNA and Reduces the Frameshifting Efficiency of Infectious Bronchitis Virus

Annexin A2 (ANXA2) is a protein implicated in diverse cellular functions, including exocytosis, DNA synthesis and cell proliferation. It was recently proposed to be involved in RNA metabolism because it was shown to associate with some cellular mRNA. Here, we identified ANXA2 as a RNA binding protein (RBP) that binds IBV (Infectious Bronchitis Virus) pseudoknot RNA. We first confirmed the binding of ANXA2 to IBV pseudoknot RNA by ultraviolet crosslinking and showed its binding to RNA pseudoknot with ANXA2 protein in vitro and in the cells. Since the RNA pseudoknot located in the frameshifting region of IBV was used as bait for cellular RBPs, we tested whether ANXA2 could regulate the frameshfting of IBV pseudoknot RNA by dual luciferase assay. Overexpression of ANXA2 significantly reduced the frameshifting efficiency from IBV pseudoknot RNA and knockdown of the protein strikingly increased the frameshifting efficiency. The results suggest that ANXA2 is a cellular RBP that can modulate the frameshifting efficiency of viral RNA, enabling it to act as an anti-viral cellular protein, and hinting at roles in RNA metabolism for other cellular mRNAs.

Programmed ribosomal frameshifting in the expression of the regulator of intestinal stem cell proliferation, adenomatous polyposis coli (APC)

A programmed ribosomal frameshift (PRF) in the decoding of APC (adenomatous polyposis coli) mRNA has been identified and characterized in Caenorhabditis worms, Drosophila and mosquitoes. The frameshift product lacks the C-terminal approximately one-third of the product of standard decoding and instead has a short sequence encoded by the -1 frame which is just 13 residues in C. elegans, but is 125 in D. melanogaster. The frameshift site is A_AA.A_AA.C in Caenorhabditids, fruit flies and the mosquitoes studied while a variant A_AA.A_AA.A is found in some other nematodes. The predicted secondary RNA structure of the downstream stimulators varies considerably in the species studied. In the twelve sequenced Drosophila genomes, it is a long stem with a four-way junction in its loop. In the five sequenced Caenorhabditis species, it is a short RNA pseudoknot with an additional stem in loop 1. The efficiency of frameshifting varies significantly, depending on the particular stimulator within the frameshift cassette, when tested with reporter constructs in rabbit reticulocyte lysates. Phylogenetic analysis of the distribution of APC programmed ribosomal frameshifting cassettes suggests it has an ancient origin and raises questions about a possibility of synthesis of alternative protein products during expression of APC in other organisms such as humans. The origin of APC as a PRF candidate emerged from a prior study of evolutionary signatures derived from comparative analysis of the 12 fly genomes. Three other proposed PRF candidates (Xbp1, CG32736, CG14047) with switches in conservation of reading frames are likely explained by mechanisms other than PRF.

Evolution of protease inhibitor resistance in the gag and pol genes of HIV subtype G isolates

To analyse HIV Gag cleavage site (CS) and non-CS mutations in HIV non-B isolates from patients failing antiretroviral therapy. Twenty-one HIV isolates were obtained from patients infected with HIV subtype G during an outbreak in Russia 20 years ago. Most patients were failing antiretroviral therapy when genotyping was performed. HIV Gag CS mutations accumulated in protease inhibitor (PI)-resistant HIV isolates and were correlated with the presence of three or more PI resistance mutations. Only 1 of 11 HIV isolates carrying major protease mutations did not harbour treatment-associated CS mutations. Natural polymorphism 453T, often found in HIV non-B subtypes, seems to favour the selection of CS mutation 453I rather than treatment-associated CS mutation 453L. Resistance-associated non-CS mutations (123E and 200I) were also observed in PI-resistant clinical isolates. Non-CS mutations in the frameshift-regulating site, which controls the synthesis of Gag-Pol, did not affect frameshift efficiency in dual luciferase assays. Of note, one of four HIV isolates from patients failing PI therapies without protease mutations harboured Gag mutations associated with PI resistance (123E and 436R) and reverse transcriptase inhibitor mutations conferring resistance to the backbone drug. HIV Gag CS mutations commonly occurred in HIV isolates from patients failing PI therapies and natural polymorphisms at the same position influence their emergence. Non-CS mutations previously associated with PI resistance were also observed in clinical isolates. Gag mutations might indicate the evolution of PI resistance even in the absence of protease mutations.

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

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

Frameshifting in Alphaviruses: A Diversity of 3′ Stimulatory Structures

Programmed ribosomal frameshifting allows the synthesis of alternative, N-terminally coincident, C-terminally distinct proteins from the same RNA. Many viruses utilize frameshifting to optimize the coding potential of compact genomes, to circumvent the host cell's canonical rule of one functional protein per mRNA, or to express alternative proteins in a fixed ratio. Programmed frameshifting is also used in the decoding of a small number of cellular genes. Recently, specific ribosomal −1 frameshifting was discovered at a conserved U_UUU_UUA motif within the sequence encoding the alphavirus 6K protein. In this case, frameshifting results in the synthesis of an additional protein, termed TF (TransFrame). This new case of frameshifting is unusual in that the −1 frame ORF is very short and completely embedded within the sequence encoding the overlapping polyprotein.The present work shows that there is remarkable diversity in the 3′ sequences that are functionally important for efficient frameshifting at the U_UUU_UUA motif. While many alphavirus species utilize a 3′ RNA structure such as a hairpin or pseudoknot, some species (such as Semliki Forest virus) apparently lack any intra-mRNA stimulatory structure, yet just 20 nt 3′-adjacent to the shift site stimulates up to 10% frameshifting. The analysis, both experimental and bioinformatic, significantly expands the known repertoire of −1 frameshifting stimulators in mammalian and insect systems.

Evidence for ribosomal frameshifting and a novel overlapping gene in the genomes of insect-specific flaviviruses

Flaviviruses have a positive-sense, single-stranded RNA genome of ∼11 kb, encoding a large polyprotein that is cleaved to produce ∼10 mature proteins. Cell fusing agent virus, Kamiti River virus, Culex flavivirus and several recently discovered flaviviruses have no known vertebrate host and apparently infect only insects. We present compelling bioinformatic evidence for a 253–295 codon overlapping gene (designated fifo) conserved throughout these insect-specific flaviviruses and immunofluorescent detection of its product. Fifo overlaps the NS2A/NS2B coding sequence in the − 1/+ 2 reading frame and is most likely expressed as a trans-frame fusion protein via ribosomal frameshifting at a conserved GGAUUUY slippery heptanucleotide with 3′-adjacent RNA secondary structure (which stimulates efficient frameshifting in vitro). The discovery bears striking parallels to the recently discovered ribosomal frameshifting site in the NS2A coding sequence of the Japanese encephalitis serogroup of flaviviruses and suggests that programmed ribosomal frameshifting may be more widespread in flaviviruses than currently realized.

Interaction of the HIV-1 frameshift signal with the ribosome

Ribosomal frameshifting on viral RNAs relies on the mechanical properties of structural elements, often pseudoknots and more rarely stem-loops, that are unfolded by the ribosome during translation. In human immunodeficiency virus (HIV)-1 type B a long hairpin containing a three-nucleotide bulge is responsible for efficient frameshifting. This three-nucleotide bulge separates the hairpin in two domains: an unstable lower stem followed by a GC-rich upper stem. Toeprinting and chemical probing assays suggest that a hairpin-like structure is retained when ribosomes, initially bound at the slippery sequence, were allowed multiple EF-G catalyzed translocation cycles. However, while the upper stem remains intact the lower stem readily melts. After the first, and single step of translocation of deacylated tRNA to the 30 S P site, movement of the mRNA stem-loop in the 5′ direction is halted, which is consistent with the notion that the downstream secondary structure resists unfolding. Mechanical stretching of the hairpin using optical tweezers only allows clear identification of unfolding of the upper stem at a force of 12.8 ± 1.0 pN. This suggests that the lower stem is unstable and may indeed readily unfold in the presence of a translocating ribosome.

Triplex structures in an RNA pseudoknot enhance mechanical stability and increase efficiency of –1 ribosomal frameshifting

Many viruses use programmed -1 ribosomal frameshifting to express defined ratios of structural and enzymatic proteins. Pseudoknot structures in messenger RNAs stimulate frameshifting in upstream slippery sequences. The detailed molecular determinants of pseudoknot mechanical stability and frameshifting efficiency are not well understood. Here we use single-molecule unfolding studies by optical tweezers, and frameshifting assays to elucidate how mechanical stability of a pseudoknot and its frameshifting efficiency are regulated by tertiary stem-loop interactions. Mechanical unfolding of a model pseudoknot and mutants designed to dissect specific interactions reveals that mechanical stability depends strongly on triplex structures formed by stem-loop interactions. Combining single-molecule and mutational studies facilitates the identification of pseudoknot folding intermediates. Average unfolding forces of the pseudoknot and mutants ranging from 50 to 22 picoNewtons correlated with frameshifting efficiencies ranging from 53% to 0%. Formation of major-groove and minor-groove triplex structures enhances pseudoknot stem stability and torsional resistance, and may thereby stimulate frameshifting. Better understanding of the molecular determinants of frameshifting efficiency may facilitate the development of anti-virus therapeutics targeting frameshifting.

Glucose Signaling Pathway and Growth Conditions Regulate Gene Expression in Retrotransposon Ty2

Gene expression in the yeast retrotransposon Ty2 is regulated at transcriptional and translational levels. In this study, we have shown that the transcription of Ty2 is partially dependent on the membrane-bound glucose sensors Gpr1p and Mth1p in Saccharomyces cerevisiae. Transcription of Ty2 decreased approx. 3-fold in the gpr1, mth1 yeast mutant. Moreover, our results revealed that the transcription of Ty2 fluctuates during the growth stages of S. cerevisae. Both transcription and the frameshift rate of Ty2 rapidly dropped when the stationary stage yeast cells were inoculated into fresh medium. There was an instant activation of Ty2 transcription and a high level expression during the entire logarithmic stage of yeast growth. However, the transcription of Ty2 decreased 2-fold when the yeast cultures entered the stationary stage. The frameshift rate in Ty2 also varied depending on the growth conditions. The highest frameshift level was observed during the mid-logarithmic stage. It decreased up to 2-fold during the stationary stage. Furthermore, we have found that the frameshift rate of Ty2 diminished at least 5-fold in slowly growing yeasts. These results indicate that the transcription and the frameshift efficiency are coordinately regulated in the retrotransposon Ty2 depending on the growth conditions of S. cerevisiae.

Increased -1 ribosomal frameshifting efficiency by yeast prion-like phenotype [ PSI+

Programmed -1 ribosomal frameshifting (-1RF) is usually regulated by a slippery sequence and an RNA secondary structure but can be affected by the slippery sequence combined with translational perturbation. Dramatic increases of -1RF efficiencies arise from the slippery sequences in [ PSI+], an epigenetic modifier of translation termination fidelity. Curing of [ PSI+] abolished such increases of -1RF efficiency. Enhanced -1RF frequency at the slippery sequences could be another physiological effect induced directly or indirectly by the perturbation of the translation process in [ PSI+] cells.

Functional Consequences of Human Immunodeficiency Virus Escape from an HLA-B*13-Restricted CD8 + T-Cell Epitope in p1 Gag Protein

The observed association between HLA-B*13 and control of human immunodeficiency virus type 1 (HIV-1) infection has been linked to the number of Gag-specific HLA-B*13-restricted cytotoxic T-cell (CTL) responses identified. To date, the Gag escape mutations described that result in an in vitro fitness cost to the virus have been located within structural protein p24 only. Here we investigated the hypothesis that CTL escape mutations within other regions of HIV Gag may also reduce viral fitness and contribute to immune control. We analyzed an HLA-B*13-restricted CTL response toward an epitope in p1 Gag, RQANFLGKI(429-437) (RI9), where amino acid variation at Gag residues 436 and 437 is associated with HLA-B*13 expression. In this work, we assessed the impact of amino acid substitutions at these positions on CTL recognition and on HIV-1 fitness. We demonstrated that substitutions I437L and I437M largely abrogate CTL recognition and reduce viral fitness while variants K436R and I437V have only a marginal effect on recognition and fitness. Examination of the patterns of protein synthesis indicated that the loss of fitness in the I437L and I437M mutants is associated with the accumulation of unprocessed Gag precursors. A significant reduction in ribosomal frameshifting efficiency was observed with I437M, suggesting that this mechanism contributes to the observed reduced fitness of this virus. These studies illustrate the apparent trade-off available to the virus between evasion of CTL recognition in p1 Gag and the functional consequences for viral fitness.

Selection of peptides interfering with a ribosomal frameshift in the human immunodeficiency virus type 1.

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.

Discovery of frameshifting in Alphavirus 6K resolves a 20-year enigma

BackgroundThe genus Alphavirus includes several potentially lethal human viruses. Additionally, species such as Sindbis virus and Semliki Forest virus are important vectors for gene therapy, vaccination and cancer research, and important models for virion assembly and structural analyses. The genome encodes nine known proteins, including the small '6K' protein. 6K appears to be involved in envelope protein processing, membrane permeabilization, virion assembly and virus budding. In protein gels, 6K migrates as a doublet – a result that, to date, has been attributed to differing degrees of acylation. Nonetheless, despite many years of research, its role is still relatively poorly understood.ResultsWe report that ribosomal -1 frameshifting, with an estimated efficiency of ~10–18%, occurs at a conserved UUUUUUA motif within the sequence encoding 6K, resulting in the synthesis of an additional protein, termed TF (TransFrame protein; ~8 kDa), in which the C-terminal amino acids are encoded by the -1 frame. The presence of TF in the Semliki Forest virion was confirmed by mass spectrometry. The expression patterns of TF and 6K were studied by pulse-chase labelling, immunoprecipitation and immunofluorescence, using both wild-type virus and a TF knockout mutant. We show that it is predominantly TF that is incorporated into the virion, not 6K as previously believed. Investigation of the 3' stimulatory signals responsible for efficient frameshifting at the UUUUUUA motif revealed a remarkable diversity of signals between different alphavirus species.ConclusionOur results provide a surprising new explanation for the 6K doublet, demand a fundamental reinterpretation of existing data on the alphavirus 6K protein, and open the way for future progress in the further characterization of the 6K and TF proteins. The results have implications for alphavirus biology, virion structure, viroporins, ribosomal frameshifting, and bioinformatic identification of novel frameshift-expressed genes, both in viruses and in cellular organisms.