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Treatment of HIV-1 has largely involved targeting viral enzymes using a cocktail of inhibitors. However, resistance to these inhibitors and toxicity in the long term have pushed the field to identify new therapeutic targets. To that end, -1 programmed ribosomal frameshifting (-1 PRF) has gained attention as a potential node for therapeutic intervention. In this process, a ribosome moves one nucleotide backward in the course of translating a mRNA, revealing a new reading frame for protein synthesis. In HIV-1, -1 PRF allows the virus to regulate the ratios of enzymatic and structural proteins as needed for correct viral particle assembly. Two RNA structural elements are central to -1 PRF in HIV: a slippery sequence and a highly conserved stable hairpin called the HIV-1 frameshifting stimulatory signal (FSS). Dysregulation of -1 PRF is deleterious for the virus. Thus, -1 PRF is an attractive target for new antiviral development. It is important to note that HIV-1 is not the only virus exploiting -1 PRF for regulating production of its proteins. Coronaviruses, including the COVID-19 pandemic virus SARS-CoV-2, also rely on -1 PRF. In SARS-CoV-2 and other coronaviruses, -1 PRF is required for synthesis of RNA-dependent RNA polymerase and several other nonstructural proteins. Coronaviruses employ a more complex RNA structural element for regulating -1 PRF called a pseudoknot. The purpose of this Account is primarily to review the development of molecules targeting HIV-1 -1 PRF. These approaches are case studies illustrating how the entire pipeline from screening to the generation of high-affinity leads might be implemented. We consider both target-based and function-based screening, with a particular focus on our group's approach beginning with a resin-bound dynamic combinatorial library (RBDCL) screen. We then used rational design approaches to optimize binding affinity, selectivity, and cellular bioavailability. Our tactic is, to the best of our knowledge, the only study resulting in compounds that bind specifically to the HIV-1 FSS RNA and reduce infectivity of laboratory and drug-resistant strains of HIV-1 in human cells. Lessons learned from strategies targeting -1 PRF HIV-1 might provide solutions in the development of antivirals in areas of unmet medical need. This includes the development of new frameshift-altering therapies for SARS-CoV-2, approaches to which are very recently beginning to appear.

The programmed frameshift element (PFE) rerouting translation from ORF1a to ORF1b is essential for the propagation of coronaviruses. The combination of genomic features that make up PFE—the overlap between the two reading frames, a slippery sequence, as well as an ensemble of complex secondary structure elements—places severe constraints on this region as most possible nucleotide substitution may disrupt one or more of these elements. The vast amount of SARS-CoV-2 sequencing data generated within the past year provides an opportunity to assess the evolutionary dynamics of PFE in great detail. Here, we performed a comparative analysis of all available coronaviral genomic data available to date. We show that the overlap between ORF1a and ORF1b evolved as a set of discrete 7, 16, 22, 25, and 31 nucleotide stretches with a well-defined phylogenetic specificity. We further examined sequencing data from over 1,500,000 complete genomes and 55,000 raw read data sets to demonstrate exceptional conservation and detect signatures of selection within the PFE region.

Toroviruses (ToVs) are enteric pathogens and comprise three species, equine torovirus (EToV), bovine torovirus (BToV), and porcine torovirus (PToV). In this study, a novel torovirus (antelope torovirus, AToV) was discovered from fecal samples of Tibetan antelopes (Pantholops hodgsonii) with viral loads of 2.10×109 to 1.76×1010 copies/g. The genome of AToV is 28,438 nucleotides (nt) in length encoding six open reading frames (ORFs) with 11 conserved domains in pp1ab and a putative slippery sequence (14171UUUAAAC14177) in the overlapping region of ORF1a and ORF1b. Phylogenetic analysis illustrated strains of AToV form a unique clade within ToVs and comparative analysis showed AToV share relatively low sequence identity with other ToVs in six ORFs (68.2–91.6% nucleotide identity). These data suggested that AToV represents a novel and distinct species of ToVs. Based on the M genes, evolutionary analysis with BEAST of AToV and other ToVs led to a most recent common ancestor estimate of 366years ago. Remarkably, recombination analysis revealed AToV was the unknown parental ToV that once involving in the recombinant events of HE genes of two Dutch strains of BToV (B150 and B155), which indicated that AToV occurred cross-species transmission and existed both in the Netherlands and China. This study revealed a novel torovirus, a natural reservoir host (Tibetan antelope) of toroviruses for the first time, and appealed to further related studies to better understand the diversity of toroviruses.

−1 programmed ribosomal frameshifting (−1 PRF) is a translational recoding mechanism used by many viral and cellular mRNAs. −1 PRF occurs at a heptanucleotide slippery sequence and is stimulated by a downstream RNA structure, most often in the form of a pseudoknot. The utilization of −1 PRF to produce proteins encoded by the −1 reading frame is wide-spread in RNA viruses, but relatively rare in cellular mRNAs. In human, only three such cases of −1 PRF events have been reported, all involving retroviral-like genes and protein products. To evaluate the extent of −1 PRF utilization in the human transcriptome, we have developed a computational scheme for identifying putative pseudoknot-dependent −1 PRF events and applied the method to a collection of 43,191 human mRNAs in the NCBI RefSeq database. In addition to the three reported cases, our study identified more than two dozen putative −1 PRF cases. The genes involved in these cases are genuine cellular genes without a viral origin. Moreover, in more than half of these cases, the frameshift site locates far upstream (>250 nt) from the stop codon of the 0 reading frame, which is nonviral-like. Using dual luciferase assays in HEK293T cells, we confirmed that the −1 PRF signals in the mRNAs of CDK5R2 and SEMA6C are functional in inducing efficient frameshifting. Our findings have significant implications in expanding the repertoire of the −1 PRF phenomenon and the protein-coding capacity of the human transcriptome.

Use of antisense oligonucleotides of the type 3'-(N)x-AAAUUUG-(N)x-5' against slippery sequence and polynucleotides against pseudoknots forming sequences of SARS-CoV-2 RNA would block the first translation of ORF1a and ORF1b and hence dwindle the virus replication. It is easy to synthesize and deliver the antisense oligonucleotides to the target by directly injecting the nano formulation into the blood.

CoV-2 which is the causative agent of COVID-19 belongs to genus betacoronaviruses. The sequence analysis of S protein of CoV-2 has shown that it has acquired a 'polybasic cleavage site' consisting of 12 aminoacids that has been predicted to enable its cleavage by other cellular proteases possibly increasing its transmissibility. The aminoacids present in receptor binding domain of S protein of SARS CoV which are critical for its binding to cellular receptor are different in CoV-2. The presence of heptanucleotide slippery sequence in ORF1 resulting in ribosomal frameshifting, and presence of transcription regulatory sequences between ORFs resulting in discontinuous transcription, are peculiar features of Coronavirus infection cycle. The exonuclease activity of nsp14 provides possible proofreading ability to RNA polymerase makes coronaviruses different from other RNA viruses allowing coronaviruses to maintain their relatively large genome size. This mini-review summarizes the peculiar features of Coronaviruses genome and the critical events during the infection cycle with focus on CoV-2.

Programmed −1 ribosomal frameshifts (−1 PRFs) are commonly used by viruses to regulate their enzymatic and structural protein levels. Human T-cell leukemia virus type 1 (HTLV-1) is a carcinogenic retrovirus that uses two independent −1 PRFs to express viral enzymes critical to establishing new HTLV-1 infections. How the cis-acting RNA elements in this viral transcript function to induce frameshifting is unknown. The objective of this work was to conclusively define the 3′ boundary of and the RNA elements within the HTLV-1 pro-pol frameshift site. We hypothesized that the frameshift site structure was a pseudoknot and that its 3′ boundary would be defined by the pseudoknot's 3′ end. To test these hypotheses, the in vitro frameshift efficiencies of three HTLV-1 pro-pol frameshift sites with different 3′ boundaries were quantified. The results indicated that nucleotides included in the longest construct were essential to highly efficient frameshift stimulation. Interestingly, only this construct could form the putative frameshift site pseudoknot. Next, the secondary structure of this frameshift site was determined. The dominant structure was an H-type pseudoknot which, together with the slippery sequence, stimulated frameshifting to 19.4(±0.3)%. The pseudoknot's critical role in frameshift stimulation was directly revealed by examining the impact of structural changes on HTLV-1 pro-pol −1 PRF. As predicted, mutations that occluded pseudoknot formation drastically reduced the frameshift efficiency. These results are significant because they demonstrate that a pseudoknot is important to HTLV-1 pro-pol −1 PRF and define the frameshift site's 3′ boundary.

The variety of structures available to an individual RNA molecule through Watson/Crick and nonclassical interactions causes them to be conformationally dynamic; that is, any single RNA may exist in and transition among multiple configurations. Recently, single-molecule methodologies have made it possible to determine the number of RNA structures present in a sample and their relative distributions, folding pathways, and conformational dynamics. In PNAS, Halma et al. (1) used single-molecule force spectroscopy (SMFS), or “molecular tweezers,” to dissect the dynamic folding and unfolding pathways of a sequence in the West Nile virus (WNV) messenger RNA (mRNA) that directs ribosomes to shift reading frame with extremely high frequency. In addition to structures identified by prior studies (2, 3), they find metastable intermediates and variants that fold along 2 mutually exclusive pathways. The finding that transitions between different conformations are maximized in the range of forces applied by elongating ribosomes makes a significant contribution to our understanding of how RNA structural dynamics can influence biological function. Structural elements in mRNAs present both problems and opportunities to ribosomes. They can act as “roadblocks,” causing elongating ribosomes to arrest. If the goal is to translate the entire mRNA, such barriers are not desirable, and ribosomes that are stopped for too long are rescued at the expense of the mRNA (4). However, if mRNA structural elements are resolvable, they can have regulatory functions by virtue of altering the kinetic parameters of translation elongation, for example, by adding time for recruitment of trans -acting factors or enabling noncanonical elongation events to proceed. One such event, called programmed −1 ribosomal frameshifting (−1 PRF), capitalizes on the ability of mRNA structures to pause ribosomes over a heptameric “slippery sequence,” whereupon a fraction of them can slip one nucleotide in the 5′ (or −1) direction (5). The elements that … [↵][1]1Email: dinman{at}umd.edu. [1]: #xref-corresp-1-1

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., 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.

A bacilliform virus was isolated from yellow catfish in China. This virus can directly adapt in cultures of EPC cells. The virus particles, which were rod-shaped approximately 120 nm long and 20 nm wide, were visible in the cytoplasm of EPC cells. The full-length genome of this virus is 26,985 nt. The genome contains four open reading frames that encode polyprotein1ab, spike glycoprotein, M protein, and N protein. There was a putative slippery sequence 14861UUUAAAC14867, which could be modeled into an RNA pseudoknot structure. The predicted amino acid sequence of pp1ab, S, M, and N genes shares 8.7%–40.2% homology with those of the two known Bafinivirus strains—WBV and FHMNV. Based on the viral morphology, genome organization, and sequence homology, this newly identified bacilliform virus appears to be Piscanivirus. To the best of our knowledge, this is the first report of Piscanivirus in yellow catfish and Piscanivirus in China.

Translational control through programmed ribosomal frameshifting (PRF) is exploited widely by viruses and increasingly documented in cellular genes. Frameshifting is induced by mRNA secondary structures that compromise ribosome fidelity during decoding of a heptanucleotide ‘slippery’ sequence. The nsp2 PRF signal of porcine reproductive and respiratory syndrome virus is distinctive in directing both −2 and −1 PRF and in its requirement for a trans-acting protein factor, the viral replicase subunit nsp1β. Here we show that the the trans-activation of frameshifting is carried out by a protein complex composed of nsp1β and a cellular poly(C) binding protein (PCBP). From the results of in vitro translation and electrophoretic mobility shift assays, we demonstrate that a PCBP/nsp1β complex binds to a C-rich sequence downstream of the slippery sequence and here mimics the activity of a structured mRNA stimulator of PRF. This is the first description of a role for a trans-acting cellular protein in PRF. The discovery broadens the repertoire of activities associated with poly(C) binding proteins and prototypes a new class of virus–host interactions.

Programmed –1 translational frameshifting is a process where the translating ribosome shifts the reading frame, which is directed by at least two stimulatory elements in the mRNA—a slippery sequence and a downstream secondary structure. Despite a lot of theoretical and experimental studies, the detailed pathway and mechanism of the –1 frameshifting remain unclear. Here, in order to understand the pathway and mechanism we consider two models to study the kinetics of the –1 frameshifting, providing quantitative explanations of the recent biochemical data of Caliskan et al. (Cell 2014, 157, 1619–1631). One model is modified from that proposed by Caliskan et al. and the other is modified from that proposed in the previous work to explain the single-molecule experimental data. It is shown that by adjusting values of some fundamental parameters both models can give quantitative explanations of the biochemical data of Caliskan et al. on the kinetics of EF-G binding and dissociation and on the kinetics of movement of tRNAs inside the ribosome. However, for the former model some adjusted parameter values deviate significantly from those determined from the available single-molecule experiments, while for the latter model all parameter values are consistent with the available biochemical and single-molecule experimental data. Thus, the latter model most likely reflects the pathway and mechanism of the –1 frameshifting., Highlights • Kinetics of EF-G binding and dissociation associated with –1 FS is studied. • Kinetics of tRNA movement associated with –1 FS is studied. • Available biochemical and single-molecule experimental data on –1 FS are explained consistently.

It has been characterized that the programmed ribosomal −1 frameshifting often occurs at the slippery sequence on the presence of a downstream mRNA pseudoknot. In some prokaryotic cases such as the dnaX gene of Escherichia coli, an additional stimulatory signal—an upstream, internal Shine–Dalgarno (SD) sequence—is also necessary to stimulate the efficient −1 frameshifting. However, the molecular and physical mechanism of the −1 frameshifting is poorly understood. Here, we propose a model of the pathway of the −1 translational frameshifting during ribosome translation of the dnaX −1 frameshift mRNA. With the model, the single-molecule fluorescence data (Chen et al. (2014) [29]) on the dynamics of the shunt either to long pausing or to normal translation, the tRNA transit and sampling dynamics in the long-paused rotated state, the EF-G sampling dynamics, the mean rotated-state lifetimes, etc., are explained quantitatively. Moreover, the model is also consistent with the experimental data (Yan et al. (2015) [30]) on translocation excursions and broad branching of frameshifting pathways. In addition, we present some predicted results, which can be easily tested by future optical trapping experiments., Highlights • A model of −1 FS is proposed. • Long pausing associated with −1 FS is explained and studied. • tRNA transit and sampling dynamics in the long-paused rotated state is studied. • EF-G sampling dynamics in the long-paused rotated state is studied. • Mean rotated-state lifetimes are studied.

Many pathogenic viruses use programmed −1 ribosomal frameshifting to regulate translation of their structural and enzymatic proteins from polycistronic mRNAs. Frameshifting is commonly stimulated by a pseudoknot located downstream from a slippery sequence, the latter positioned at the ribosomal A and P sites. We report here the structures of two crystal forms of the frameshifting RNA pseudoknot from beet western yellow virus at resolutions of 1.25 and 2.85 Å. Because of the very high resolution of 1.25 Å, ten mono- and divalent metal ions per asymmetric unit could be identified, giving insight into potential roles of metal ions in stabilizing the pseudoknot. A magnesium ion located at the junction of the two pseudoknot stems appears to play a crucial role in stabilizing the structure. Because the two crystal forms exhibit mostly unrelated packing interactions and local crystallographic disorder in the high-resolution form was resolvable, the two structures offer the most detailed view yet of the conformational preference and flexibility of an RNA pseudoknot.

Highlights • –1PRF occurs when ribosomes move over a slippery sequence. • A frameshifting pseudoknot/stem-loop element stalls ribosomes in a metastable state. • –1PRF may contribute to the quality-control machinery in eukaryotes. • Trans-acting factors (proteins, miRNAs, or antibiotics) can modulate –1PRF., Programmed −1 ribosomal frameshifting (−1PRF) is an mRNA recoding event commonly utilized by viruses and bacteria to increase the information content of their genomes. Recent results have implicated −1PRF in quality control of mRNA and DNA stability in eukaryotes. Biophysical experiments demonstrated that the ribosome changes the reading frame while attempting to move over a slippery sequence of the mRNA – when a roadblock formed by a folded downstream segment in the mRNA stalls the ribosome in a metastable conformational state. The efficiency of −1PRF is modulated not only by cis-regulatory elements in the mRNA but also by trans-acting factors such as proteins, miRNAs, and antibiotics. These recent results suggest a molecular mechanism and new important cellular roles for −1PRF.

Summary Programmed ribosomal frameshifting produces alternative proteins from a single transcript. -1-frameshifting occurs on Escherichia coli’s dnaX mRNA containing a slippery sequence AAAAAAG and peripheral mRNA structural barriers. Here we reveal hidden aspects of the frameshifting process, including its exact location on the mRNA and its timing within the translation cycle. Mass spectrometry of translated products shows that ribosomes enter the -1-frame from not one specific codon but various codons along the slippery sequence and slip by not just -1 but also -4, or +2-nucleotides. Single-ribosome translation trajectories detect distinctive codon-scale fluctuations in ribosome-mRNA displacement across the slippery sequence, representing multiple ribosomal translocation attempts during frameshifting. Flanking mRNA structural barriers mechanically stimulate the ribosome to undergo back-and-forth translocation excursions, broadly exploring reading frames. Both experiments reveal aborted translation around mutant slippery sequences, indicating that subsequent fidelity checks on newly adopted codon position base-pairings lead to either resumed translation or early termination.

Programmed -1 ribosomal frameshifting (-1PRF) is a recoding mechanism to make alternative proteins from a single mRNA transcript. -1PRF is stimulated by cis-acting signals in mRNA, a seven-nucleotide slippery sequence and a downstream secondary structure element, which is often a pseudoknot. In this study we engineered the frameshifting pseudoknot from the mouse mammary tumor virus to respond to a rationally designed small molecule naphthyridine carbamate tetramer (NCTn). We demonstrate that NCTn can stabilize the pseudoknot structure in mRNA and activate -1PRF both in vitro and in human cells. The results illustrate how NCTn-inducible -1PRF may serve as an important component of the synthetic biology toolbox for the precise control of gene expression using small synthetic molecules.

Minus-one programmed ribosomal frameshifting (-1 PRF) allows the precise maintenance of the ratio between viral proteins and is involved in the regulation of the half-lives of cellular mRNAs. Minus-one ribosomal frameshifting is activated by several stimulatory elements such as a heptameric slippery sequence (X XXY YYZ) and an mRNA secondary structure (hairpin or pseudoknot) that is positioned 2-8 nucleotides downstream from the slippery site. Upon -1 RF, the ribosomal reading frame is shifted from the normal zero frame to the -1 frame with the heptameric slippery sequence decoded as XXX YYY Z instead of X XXY YYZ. Our research group has developed chemically modified peptide nucleic acid (PNA) L and Q monomers to recognize G-C and C-G Watson-Crick base pairs, respectively, through major-groove parallel PNA·RNA-RNA triplex formation. L- and Q-incorporated PNAs show selective binding to double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). The sequence specificity and structural selectivity of L- and Q-modified PNAs may allow the precise targeting of desired viral and cellular RNA structures, and thus may serve as valuable biological tools for mechanistic studies and potential therapeutics for fighting diseases. Here, for the first time, we demonstrate by cell-free in vitro translation assays using rabbit reticulocyte lysate that the dsRNA-specific chemically modified PNAs targeting model mRNA hairpins stimulate -1 RF (from 2% to 32%). An unmodified control PNA, however, shows nonspecific inhibition of translation. Our results suggest that the modified dsRNA-binding PNAs may be advantageous for targeting structured RNAs.

Summary Ribosome frameshifting during translation of bacterial dnaX can proceed via different routes, generating a variety of distinct polypeptides. Using kinetic experiments, we show that –1 frameshifting predominantly occurs during translocation of two tRNAs bound to the slippery sequence codons. This pathway depends on a stem-loop mRNA structure downstream of the slippery sequence and operates when aminoacyl-tRNAs are abundant. However, when aminoacyl-tRNAs are in short supply, the ribosome switches to an alternative frameshifting pathway that is independent of a stem-loop. Ribosome stalling at a vacant 0-frame A-site codon results in slippage of the P-site peptidyl-tRNA, allowing for –1-frame decoding. When the –1-frame aminoacyl-tRNA is lacking, the ribosomes switch into –2 frame. Quantitative mass spectrometry shows that the –2-frame product is synthesized in vivo . We suggest that switching between frameshifting routes may enrich gene expression at conditions of aminoacyl-tRNA limitation.

1 programmed ribosomal frameshifting (PRF) in viruses is often stimulated by a pseudoknot downstream from the slippery sequence. At the PRF junction of HIV-1, transmissible gastroenteritis virus (TGEV), Barmah Forest virus (BFV), Fort Morgan virus (FMV), and Equine arteritis virus (EAV), we identified potential double pseudoknots in either a tandem mode or embedded mode. In viruses with tandem pseudoknots (5'PK3'PK), the slippery sequence is encompassed in the 5'PK. The ribosome needs to unwind the 5'PK to get to the slippery sequence. In HIV-1, the 3'PK and several alternative structures are mutually exclusive. Disruption of the tandem pseudoknots may enable one of the alternative structures to form as the effective frameshift stimulator. In TGEV/BFV/FMV, the 3'PK is a conventional frameshift stimulator. In all cases, the tandem pseudoknots may slow down the ribosome before it reaches the conventional PRF signals. In EAV, a compact pseudoknot is embedded within loop2 of the otherwise conventional frameshift-stimulating pseudoknot. All double pseudoknots have the potential to stack their stems coaxially. We built structural models of the HIV-1 and EAV double pseudoknots to show that both the tandem and embedded modes are feasible and reasonable. We hypothesize that the fundamental reason for the viruses to utilize coaxially stacked double pseudoknots is to increase the overall stability of the frameshift regulating structure, and avoid an ultra-stable single pseudoknot which may become a ribosomal roadblock. Our results significantly expand the repertoire of RNA structures and dynamics that may potentially involve in -1 PRF regulation.

Spontaneous changes in the reading frame of translation are rare (frequency of 10(-3) to 10(-4) per codon), but can be induced by specific features in the messenger RNA (mRNA). In the presence of mRNA secondary structures, a heptanucleotide 'slippery sequence' usually defined by the motif X XXY YYZ, and (in some prokaryotic cases) mRNA sequences that base pair with the 3' end of the 16S ribosomal rRNA (internal Shine-Dalgarno sequences), there is an increased probability that a specific programmed change of frame occurs, wherein the ribosome shifts one nucleotide backwards into an overlapping reading frame (-1 frame) and continues by translating a new sequence of amino acids. Despite extensive biochemical and genetic studies, there is no clear mechanistic description for frameshifting. Here we apply single-molecule fluorescence to track the compositional and conformational dynamics of individual ribosomes at each codon during translation of a frameshift-inducing mRNA from the dnaX gene in Escherichia coli. Ribosomes that frameshift into the -1 frame are characterized by a tenfold longer pause in elongation compared to non-frameshifted ribosomes, which translate through unperturbed. During the pause, interactions of the ribosome with the mRNA stimulatory elements uncouple EF-G catalysed translocation from normal ribosomal subunit reverse-rotation, leaving the ribosome in a non-canonical intersubunit rotated state with an exposed codon in the aminoacyl-tRNA site (A site). tRNA(Lys) sampling and accommodation to the empty A site and EF-G action either leads to the slippage of the tRNAs into the -1 frame or maintains the ribosome into the 0 frame. Our results provide a general mechanistic and conformational framework for -1 frameshifting, highlighting multiple kinetic branchpoints during elongation.

Hibiscus latent Singapore virus (HLSV) is a member of Tobamovirus and its full-length cDNA clones were constructed. The in vitro transcripts from two HLSV full-length cDNA clones, which contain a hepta-adenosine stretch (pHLSV-7A) and an octo-adenosine stretch (pHLSV-8A), are both infectious. The replication level of HLSV-7A in Nicotiana benthamiana protoplasts was 5-fold lower, as compared to that of HLSV-8A. The replicase proteins of HLSV-7A were produced through programmed −1 ribosomal frameshift (−1 PRF) and the 7A stretch was a slippery sequence for −1 PRF. Mutations to the downstream pseudoknot of 7A stretch showed that the pseudoknot was not required for the frameshift in vitro. The stretch was found to be extended to 8A after subsequent replication cycles in vivo. It is envisaged that HLSV employs the monotonous runs of A and −1 PRF to convert its 7A to 8A to reach higher replication for its survival in plants., Highlights • Successful construction of HLSV full-length cDNA clone. • First report of a programmed −1 frameshift function in Tobamovirus. • 7A stretch is the sole signal for programmed −1 frameshift in HLSV. • 7A stretch can be extended to 8A stretch during HLSV replication.

Tertiary structures formed by mRNAs impact the efficiency of the translation reaction. Ribosomal frameshift is a well-characterized recoding process that occurs during translation elongation. Pseudoknot and stem-loop structures may stimulate frameshifting by causing a translational halt at a slippery sequence. In this study, we evaluated the efficiency of an unusual -1 frameshift caused by a noncanonical RNA G-quadruplex structure in mammalian cells. The reporter gene construct consisting of a fluorescent protein and Luciferase enabled evaluation of apparent and absolute values of the -1 frameshift efficiency and revealed significant increase of the efficiency by G-quadrupex forming potential sequence. In addition, berberine, a small molecule that binds to and stabilizes G-quadruplex structures, further increased the frameshift efficiency. These results indicate that the stable G-quadruplex structure stimulates the unusual -1 frameshift and has a potential to regulate the frameshift with its ligand.

Programmed -1 ribosomal frameshifting (-1PRF) is tightly regulated by messenger RNA (mRNA) sequences and structures in expressing two or more proteins with precise ratios from a single mRNA. Using single-molecule fluorescence resonance energy transfer (smFRET) between (Cy5)EF-G and (Cy3)tRNALys, we studied the translational elongation dynamics of -1PRF in the Escherichia coli dnaX gene, which contains three frameshifting signals: a slippery sequence (A AAA AAG), a Shine-Dalgarno (SD) sequence and a downstream hairpin. The frameshift promoting signals mostly impair the EF-G-catalyzed translocation step of the two tRNALys and the slippery codons from the A- and P- sites. The hairpin acts as a road block slowing the translocation rate. The upstream SD sequence together with the hairpin promotes dissociation of futile EF-G and thus causes multiple EF-G driven translocation attempts. A slippery sequence also helps dissociation of the EF-G by providing alternative base-pairing options. These results indicate that frameshifting takes place during the repetitive ribosomal conformational changes associated with EF-G dissociation upon unsuccessful translocation attempts of the second slippage codon from the A- to the P- sites.

Background Retroviruses transcribe messenger RNA for the overlapping Gag and Gag-Pol polyproteins, by using a programmed -1 ribosomal frameshift which requires a slippery sequence and an immediate downstream stem-loop secondary structure, together called frameshift stimulating signal (FSS). It follows that the molecular evolution of this genomic region of HIV-1 is highly constrained, since the retroviral genome must contain a slippery sequence (sequence constraint), code appropriate peptides in reading frames 0 and 1 (coding requirements), and form a thermodynamically stable stem-loop secondary structure (structure requirement). Results We describe a unique computational tool, RNAsampleCDS, designed to compute the number of RNA sequences that code two (or more) peptides p,q in overlapping reading frames, that are identical (or have BLOSUM/PAM similarity that exceeds a user-specified value) to the input peptides p,q. RNAsampleCDS then samples a user-specified number of messenger RNAs that code such peptides; alternatively, RNAsampleCDS can exactly compute the position-specific scoring matrix and codon usage bias for all such RNA sequences. Our software allows the user to stipulate overlapping coding requirements for all 6 possible reading frames simultaneously, even allowing IUPAC constraints on RNA sequences and fixing GC-content. We generalize the notion of codon preference index (CPI) to overlapping reading frames, and use RNAsampleCDS to generate control sequences required in the computation of CPI. Moreover, by applying RNAsampleCDS, we are able to quantify the extent to which the overlapping coding requirement in HIV-1 [resp. HCV] contribute to the formation of the stem-loop [resp. double stem-loop] secondary structure known as the frameshift stimulating signal. Using our software, we confirm that certain experimentally determined deleterious HCV mutations occur in positions for which our software RNAsampleCDS and RNAiFold both indicate a single possible nucleotide. We generalize the notion of codon preference index (CPI) to overlapping coding regions, and use RNAsampleCDS to generate control sequences required in the computation of CPI for the Gag-Pol overlapping coding region of HIV-1. These applications show that RNAsampleCDS constitutes a unique tool in the software arsenal now available to evolutionary biologists. Conclusion Source code for the programs and additional data are available at http://bioinformatics.bc.edu/clotelab/RNAsampleCDS/. Electronic supplementary material The online version of this article (doi:10.1186/s12859-016-1389-7) contains supplementary material, which is available to authorized users.

A ribosome is a molecular motor [1] that synthesises a polypeptide using a mRNA template that also serves as a track for its motor-like directed movement in steps of three nucleotides (a codon). However, on some special “slippery” sequence of nucleotides on the mRNA the reading frame of a ribosome can shift backward by one nucleotide which is identified as a −1 programmed frameshift. In addition to the slippery sequence, a secondary structure of the mRNA strand (for example, a pseudo knot), located downstream from the slippery sequence, is also essential for programmed −1 frameshift. Since the shifted reading frame picks up a sequence of amino acids that is, in general, different from the sequence that would have been selected by the unshifted original reading frame, a ribosome that resumes protein synthesis after such a frameshift produces a fusion protein. Often many ribosomes simultaneously move along the same mRNA track, all synthesising identical polypeptides. The stiffness of the mRNA secondary structure determines the extent of ribosomal crowding in the mRNA segment upstream from that structure. Using a theoretical model we demonstrate how the increasing stiffness of the secondary structure of the mRNA causes increasing crowding of the ribosomes that, in turn, leads to a non-monotonic variation of the efficiency of frameshift [3]. Our numerical estimates indicate that the novel phenomenon predicted by our theoretical model can be tested experimentally by ribosome profiling technique [4].[1] D. Chowdhury, Biophys. J. 104, 2331 (2013).[2] J.F. Atkins and R.F. Gesteland (eds.) Recoding: Expansion of Decoding Rules Enriches Gene Expression, (Springer, 2010).[3] B. Mishra and D. Chowdhury, to be published (2015).[4] N.T. Ingolia, Nat. Rev. Genet. 15, 205 (2014).

1 Programmed ribosomal frameshifting is a translational recoding event in which ribosomes slip backward along messenger RNA presumably due to increased tension disrupting the codon-anticodon interaction at the ribosome's coding site. Single-molecule physical methods and recent experiments characterizing the physical properties of mRNA's slippery sequence as well as the mechanical stability of downstream mRNA structure motifs that give rise to frameshifting are discussed. Progress in technology, experimental assays, and data analysis methods hold promise for accurate physical modeling and quantitative understanding of -1 programmed ribosomal frameshifting.

We develop a stochastic model for the programmed frameshift of ribosomes synthesizing a protein while moving along a mRNA template. Normally the reading frame of a ribosome decodes successive triplets of nucleotides on the mRNA in a step-by-step manner. We focus on the programmed shift of the ribosomal reading frame, forward or backward, by only one nucleotide which results in a fusion protein; it occurs when a ribosome temporarily loses its grip to its mRNA track. Special "slippery" sequences of nucleotides and also downstream secondary structures of the mRNA strand are believed to play key roles in programmed frameshift. Here we explore the role of an hitherto neglected parameter in regulating -1 programmed frameshift. Specifically, we demonstrate that the frameshift frequency can be strongly regulated also by the density of the ribosomes, all of which are engaged in simultaneous translation of the same mRNA, at and around the slippery sequence. Monte Carlo simulations support the analytical predictions obtained from a mean-field analysis of the stochastic dynamics., Comment: This is an author-created, un-copyedited final version of an article published in EPL

Programmed −1 ribosomal frameshifting is widely used in the expression of RNA virus replicases and represents a potential target for antiviral intervention. There is interest in determining the extent to which frameshifting efficiency can be modulated before virus replication is compromised, and we have addressed this question using the alpharetrovirus Rous sarcoma virus (RSV) as a model system. In RSV, frameshifting is essential in the production of the Gag-Pol polyprotein from the overlapping gag and pol coding sequences. The frameshift signal is composed of two elements, a heptanucleotide slippery sequence and, just downstream, a stimulatory RNA structure that has been proposed to be an RNA pseudoknot. Point mutations were introduced into the frameshift signal of an infectious RSV clone, and virus replication was monitored following transfection and subsequent infection of susceptible cells. The introduced mutations were designed to generate a range of frameshifting efficiencies, yet with minimal impact on encoded amino acids. Our results reveal that point mutations leading to a 3-fold decrease in frameshifting efficiency noticeably reduce virus replication and that further reduction is severely inhibitory. In contrast, a 3-fold stimulation of frameshifting is well tolerated. These observations suggest that small-molecule inhibitors of frameshifting are likely to have potential as agents for antiviral intervention. During the course of this work, we were able to confirm, for the first time in vivo , that the RSV stimulatory RNA is indeed an RNA pseudoknot but that the pseudoknot per se is not absolutely required for virus viability.

Programmed -1 ribosomal frameshifting (-1RFS) is one of well-known alternative decoding mechanisms found in nature. -1RFS mechanism has been first described as a mechanism that controls the relative expression levels of two proteins in metazoan viruses. 1 Furthermore, among many retroviruses, plant viruses, coronaviruses and certain bacterial and protozoan genes, this has been identified as a mechanism that modulates the translation of two proteins encoded by overlapping open reading frames present in one mRNA. 2,3 Their prevalence across such an evolutionarily diverse distribution manifests that such sites have evolved several times. Sequence comparison and molecular genetic analysis of many of -1RFS sites show a canonical structure for these frameshift sites. It is now clear that two cis-elements in mRNA alone are enough to create this unusual alternative decoding event, although there are some possible influences of other trans-factors on frameshifting efficiency. Two ciselements are involved in this alternative reading process of genetic codes during translation. 4 One is the slippery sequence, a heptanucleotide motif XXXYYYN, where -1 frameshifting occurs. The other is a downstream RNA secondary structure, usually a pseudoknot. The -1RFS system containing these two cis-elements can induce -1 frameshifting at a slippery site (slippery sequence) with 1 to over 30% of efficiency. Recent studies have worked on not only the detailed mechanism and functional importance of -1RFS, 5-7 but

Programmed ribosomal frameshifting is a translational recoding mechanism commonly used by RNA viruses to express two or more proteins from a single mRNA at a fixed ratio. An essential element in this process is the presence of an RNA secondary structure, such as a pseudoknot or a hairpin, located downstream of the slippery sequence. Here, we have tested the efficiency of RNA oligonucleotides annealing downstream of the slippery sequence to induce frameshifting in vitro. Maximal frameshifting was observed with oligonucleotides of 12–18 nt. Antisense oligonucleotides bearing locked nucleid acid (LNA) modifications also proved to be efficient frameshift-stimulators in contrast to DNA oligonucleotides. The number, sequence and location of LNA bases in an otherwise DNA oligonucleotide have to be carefully manipulated to obtain optimal levels of frameshifting. Our data favor a model in which RNA stability at the entrance of the ribosomal tunnel is the major determinant of stimulating slippage rather than a specific three-dimensional structure of the stimulating RNA element.

Simian retrovirus type-1 uses programmed ribosomal frameshifting to control expression of the Gag-Pol polyprotein from overlapping gag and pol open-reading frames. The frameshifting signal consists of a heptanucleotide slippery sequence and a downstream-located 12-base pair pseudoknot. The solution structure of this pseudoknot, previously solved by NMR [Michiels,P.J., Versleijen,A.A., Verlaan,P.W., Pleij,C.W., Hilbers,C.W. and Heus,H.A. (2001) Solution structure of the pseudoknot of SRV-1 RNA, involved in ribosomal frameshifting. J. Mol. Biol., 310, 1109–1123] has a classical H-type fold and forms an extended triple helix by interactions between loop 2 and the minor groove of stem 1 involving base–base and base–sugar contacts. A mutational analysis was performed to test the functional importance of the triple helix for −1 frameshifting in vitro. Changing bases in L2 or base pairs in S1 involved in a base triple resulted in a 2- to 5-fold decrease in frameshifting efficiency. Alterations in the length of L2 had adverse effects on frameshifting. The in vitro effects were well reproduced in vivo, although the effect of enlarging L2 was more dramatic in vivo. The putative role of refolding kinetics of frameshifter pseudoknots is discussed. Overall, the data emphasize the role of the triple helix in −1 frameshifting.

An efficient −1 programmed ribosomal frameshifting (PRF) signal requires an RNA slippery sequence and a downstream RNA stimulator, and the hairpin-type pseudoknot is the most common stimulator. However, a pseudoknot is not sufficient to promote −1 PRF. hTPK-DU177, a pseudoknot derived from human telomerase RNA, shares structural similarities with several −1 PRF pseudoknots and is used to dissect the roles of distinct structural features in the stimulator of −1 PRF. Structure-based mutagenesis on hTPK-DU177 reveals that the −1 PRF efficiency of this stimulator can be modulated by sequential removal of base–triple interactions surrounding the helical junction. Further analysis of the junction-flanking base triples indicates that specific stem–loop interactions and their relative positions to the helical junction play crucial roles for the −1 PRF activity of this pseudoknot. Intriguingly, a bimolecular pseudoknot approach based on hTPK-DU177 reveals that continuing triplex structure spanning the helical junction, lacking one of the loop-closure features embedded in pseudoknot topology, can stimulate −1 PRF. Therefore, the triplex structure is an essential determinant for the DU177 pseudoknot to stimulate −1 PRF. Furthermore, it suggests that −1 PRF, induced by an in-trans RNA via specific base–triple interactions with messenger RNAs, can be a plausible regulatory function for non-coding RNAs.

The −1 programmed ribosomal frameshifting (−1 PRF) mechanism utilized by many viruses is dependent on a heptanucleotide slippery sequence and a downstream secondary structure element. In the current study, the RNA structure downstream from the slippery site of cocksfoot mottle sobemovirus (CfMV) was proven to be a 12 bp stem-loop with a single bulge and a tetranucleotide loop. Several deletion and insertion mutants with altered stem-loop structures were tested in wheat germ extract (WGE) for frameshifting efficiency. The impact of the same mutations on virus infectivity was tested in oat plants. Mutations shortening or destabilizing the stem region reduced significantly but did not abolish −1 PRF in WGE. The same mutations proved to be deleterious for virus infection. However, extending the loop region to seven nucleotides had no significant effect on frameshifting efficiency in WGE and did not hamper virus replication in infected leaves. This is the first report about the experimentally proven RNA secondary structure directing −1 PRF of sobemoviruses.

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 approximately 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 (K(d) = 2.8 microM). This binding was confirmed and localized to the RNA using NMR. Further analysis revealed that this compound increased the RNA stability by approximately 5 degrees C and decreased translational frameshifting by 28% (+/-14%), as measured in vitro.

In the course of sobemovirus gene cloning the complete genome of Ryegrass mottle virus (RGMoV) was sequenced. Sequence analysis revealed differences including missing and extraneous nucleotides in comparison to the previously published sequence (Zhang, Toriyama, Takanashi, J. Gen. Plant Pathol. 67, 63 (2001)). A gene coding for a typical sobemovirus 3C-like serine protease was identified in ORF2a after multiple sequence alignment analysis. The newly identified 57-amino-acid stretch in ORF2a showed similarities ranging from 38.5 to 50.9% among sequenced genes of sobemovirus proteases. ORF analysis of the RGMoV polyprotein coding sequence demonstrated the arrangement of ORF2b coding for RNA-dependent RNA polymerase (RdRP) in the -1 frame in regard to ORF2a. The localization of conserved among sobemoviruses slippery sequence (UUUAAAC) at the 3'-end of ORF2a suggests the translation of RdRP via a -1 ribosomal frameshifting mechanism, allowing to include the RGMoV in the sobemovirus group with a Cocksfoot mottle virus-like (CfMV-like) genome organization.

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.

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.

Programmed ribosomal frameshifting is often used by viral pathogens including HIV. Slippery sequences present in some mRNAs cause the ribosome to shift reading frame. The resulting protein is thus encoded by one reading frame upstream from the slippery sequence and by another reading frame downstream from the slippery sequence. Although the mechanism is not well understood, frameshifting is known to be stimulated by an mRNA structure such as a pseudoknot. Here, we show that the efficiency of frameshifting relates to the mechanical strength of the pseudoknot. Two pseudoknots derived from the Infectious Bronchitis Virus were used, differing by one base pair in the first stem. In Escherichia coli , these two pseudoknots caused frameshifting frequencies that differed by a factor of two. We used optical tweezers to unfold the pseudoknots. The pseudoknot giving rise to the highest degree of frameshifting required a nearly 2-fold larger unfolding force than the other. The observed energy difference cannot be accounted for by any existing model. We propose that the degree of ribosomal frameshifting is related to the mechanical strength of RNA pseudoknots. Our observations support the “9 Å model” that predicts some physical barrier is needed to force the ribosome into the −1 frame. Also, our findings support the recent observation made by cryoelectron microscopy that mechanical interaction between a ribosome and a pseudoknot causes a deformation of the A-site tRNA. The result has implications for the understanding of genetic regulation, reading frame maintenance, tRNA movement, and unwinding of mRNA secondary structures by ribosomes.

Synthetic biology and the rational design of biological devices depend on the availability of standardized and interchangeable biological parts with diverse range of functions. Reliable access to different reading frames during translation has largely been overlooked as functionality for bioengineering applications. Here we report the construction and initial characterization of the first member of such a class of biological parts that conforms to the BioBrick Standard (RFC25), allowing its interchangeable use in biological devices. Using our standardized frameshifting signal consisting of a UUUAAAG slippery sequence, a 6 nt spacer and an engineered pseudoknot based on the infectious bronchitis virus pseudoknot PK401 embedded in a dual reporter construct, we confirm that the frameshifting activity is comparable to the previously published frequency despite the introduced sequence changes. The frameshifting activity is demonstrated using SDS-PAGE and fluorescence spectroscopy. Standardized programmable ribosomal frameshift parts with specific frameshifting frequencies will be of utility for applications such as double coding DNA sequences by expanding the codable space into the -1 frame. Programmed shifting into the -1 frame to bypass a stop codon allows labeling of a protein pool with a fixed stoichiometry of fusion protein, as well as the construction of multi-enzyme expression constructs with specific expression ratios. A detailed understanding of the structural basis of programmed frameshifting will provide the opportunities to rationally design frameshifting elements with a wide range of applications in synthetic biology, including signals that are regulated by small ligands.

Many bacteria employ a protein organelle, the carboxysome, to catalyze carbon dioxide fixation in the Calvin Cycle. Only 10 genes from Halothiobacillus neapolitanus are sufficient for heterologous expression of carboxysomes in Escherichia coli, opening the door to detailed mechanistic analysis of the assembly process of this complex (more than 200MDa). One of these genes, csoS2, has been implicated in assembly but ascribing a molecular function is confounded by the observation that the single csoS2 gene yields expression of two gene products and both display an apparent molecular weight incongruent with the predicted amino acid sequence. Here, we elucidate the co-translational mechanism responsible for the expression of the two protein isoforms. Specifically, csoS2 was found to possess -1 frameshifting elements that lead to the production of the full-length protein, CsoS2B, and a truncated protein, CsoS2A, which possesses a C-terminus translated from the alternate frame. The frameshifting elements comprise both a ribosomal slippery sequence and a 3' secondary structure, and ablation of either sequence is sufficient to eliminate the slip. Using these mutants, we investigated the individual roles of CsoS2B and CsoS2A on carboxysome formation. In this in vivo formation assay, cells expressing only the CsoS2B isoform were capable of producing intact carboxysomes, while those with only CsoS2A were not. Thus, we have answered a long-standing question about the nature of CsoS2 in this model microcompartment and demonstrate that CsoS2B is functionally distinct from CsoS2A in the assembly of α-carboxysomes.

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.

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.