Frameshift Efficiency Research Articles

An analysis by metabolic labelling of the encephalomyocarditis virus ribosomal frameshifting efficiency and stimulators.

Programmed −1 ribosomal frameshifting is a mechanism of gene expression whereby specific signals within messenger RNAs direct a proportion of ribosomes to shift −1 nt and continue translating in the new reading frame. Such frameshifting normally depends on an RNA structure stimulator 3′-adjacent to a ‘slippery’ heptanucleotide shift site sequence. Recently we identified an unusual frameshifting mechanism in encephalomyocarditis virus, where the stimulator involves a trans-acting virus protein. Thus, in contrast to other examples of −1 frameshifting, the efficiency of frameshifting in encephalomyocarditis virus is best studied in the context of virus infection. Here we use metabolic labelling to analyse the frameshifting efficiency of wild-type and mutant viruses. Confirming previous results, frameshifting depends on a G_GUU_UUU shift site sequence and a 3′-adjacent stem-loop structure, but is not appreciably affected by the ‘StopGo’ sequence present ~30 nt upstream. At late timepoints, frameshifting was estimated to be 46–76 % efficient.

Programmed Ribosomal Frameshifting Generates a Copper Transporter and a Copper Chaperone from the Same Gene

Metal efflux pumps maintain ion homeostasis in the cell. The functions of the transporters are often supported by chaperone proteins, which scavenge the metal ions from the cytoplasm. Although the copper ion transporter CopA has been known in Escherichia coli, no gene for its chaperone had been identified. We show that the CopA chaperone is expressed in E.coli from the same gene that encodes the transporter. Some ribosomes translating copA undergo programmed frameshifting, terminate translation in the -1 frame, and generate the 70 aa-long polypeptide CopA(Z), which helps cells survive toxic copper concentrations. The high efficiency of frameshifting is achieved by the combined stimulatory action of a"slippery" sequence, an mRNA pseudoknot, and the CopA nascent chain. Similar mRNA elements are not only found in the copA genes of other bacteria but are also present in ATP7B, the human homolog of copA, and direct ribosomal frameshifting invivo.

A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish.

ABSTRACTClustered regularly interspaced palindromic repeats (CRISPR)/Cas-based adaptive immunity against pathogens in bacteria has been adapted for genome editing and applied in zebrafish (Danio rerio) to generate frameshift mutations in protein-coding genes. Although there are methods to detect, quantify and sequence CRISPR/Cas9-induced mutations, identifying mutations in F1 heterozygous fish remains challenging. Additionally, sequencing a mutation and assuming that it causes a frameshift does not prove causality because of possible alternative translation start sites and potential effects of mutations on splicing. This problem is compounded by the relatively few antibodies available for zebrafish proteins, limiting validation at the protein level. To address these issues, we developed a detailed protocol to screen F1 mutation carriers, and clone and sequence identified mutations. In order to verify that mutations actually cause frameshifts, we created a fluorescent reporter system that can detect frameshift efficiency based on the cloning of wild-type and mutant cDNA fragments and their expression levels. As proof of principle, we applied this strategy to three CRISPR/Cas9-induced mutations in pycr1a, chd7 and hace1 genes. An insertion of seven nucleotides in pycr1a resulted in the first reported observation of exon skipping by CRISPR/Cas9-induced mutations in zebrafish. However, of these three mutant genes, the fluorescent reporter revealed effective frameshifting exclusively in the case of a two-nucleotide deletion in chd7, suggesting activity of alternative translation sites in the other two mutants even though pycr1a exon-skipping deletion is likely to be deleterious. This article provides a protocol for characterizing frameshift mutations in zebrafish, and highlights the importance of checking mutations at the mRNA level and verifying their effects on translation by fluorescent reporters when antibody detection of protein loss is not possible.

Mechanical unfolding kinetics of the SRV-1 gag-pro mRNA pseudoknot: possible implications for -1 ribosomal frameshifting stimulation.

Minus-one ribosomal frameshifting is a translational recoding mechanism widely utilized by many RNA viruses to generate accurate ratios of structural and catalytic proteins. An RNA pseudoknot structure located in the overlapping region of the gag and pro genes of Simian Retrovirus type 1 (SRV-1) stimulates frameshifting. However, the experimental characterization of SRV-1 pseudoknot (un)folding dynamics and the effect of the base triple formation is lacking. Here, we report the results of our single-molecule nanomanipulation using optical tweezers and theoretical simulation by steered molecular dynamics. Our results directly reveal that the energetic coupling between loop 2 and stem 1 via minor-groove base triple formation enhances the mechanical stability. The terminal base pair in stem 1 (directly in contact with a translating ribosome at the slippery site) also affects the mechanical stability of the pseudoknot. The −1 frameshifting efficiency is positively correlated with the cooperative one-step unfolding force and inversely correlated with the one-step mechanical unfolding rate at zero force. A significantly improved correlation was observed between −1 frameshifting efficiency and unfolding rate at forces of 15–35 pN, consistent with the fact that the ribosome is a force-generating molecular motor with helicase activity. No correlation was observed between thermal stability and −1 frameshifting efficiency.

Salt Effects on the Thermodynamics of a Frameshifting RNA Pseudoknot under Tension

Because of the potential link between −1 programmed ribosomal frameshifting and response of a pseudoknot (PK) RNA to force, a number of single-molecule pulling experiments have been performed on PKs to decipher the mechanism of programmed ribosomal frameshifting. Motivated in part by these experiments, we performed simulations using a coarse-grained model of RNA to describe the response of a PK over a range of mechanical forces (fs) and monovalent salt concentrations (Cs). The coarse-grained simulations quantitatively reproduce the multistep thermal melting observed in experiments, thus validating our model. The free energy changes obtained in simulations are in excellent agreement with experiments. By varying f and C, we calculated the phase diagram that shows a sequence of structural transitions, populating distinct intermediate states. As f and C are changed, the stem–loop tertiary interactions rupture first, followed by unfolding of the 3′-end hairpin (I⇌F). Finally, the 5′-end hairpin unravels, producing an extended state (E⇌I). A theoretical analysis of the phase boundaries shows that the critical force for rupture scales as (logCm)α with α=1(0.5) for E⇌I (I⇌F) transition. This relation is used to obtain the preferential ion–RNA interaction coefficient, which can be quantitatively measured in single-molecule experiments, as done previously for DNA hairpins. A by-product of our work is the suggestion that the frameshift efficiency is likely determined by the stability of the 5′-end hairpin that the ribosome first encounters during translation.

Stability of HIV Frameshift Site RNA Correlates with Frameshift Efficiency and Decreased Virus Infectivity.

Human immunodeficiency virus (HIV) replication is strongly dependent upon a programmed ribosomal frameshift. Here we investigate the relationships between the thermodynamic stability of the HIV type 1 (HIV-1) RNA frameshift site stem-loop, frameshift efficiency, and infectivity, using pseudotyped HIV-1 and HEK293T cells. The data reveal a strong correlation between frameshift efficiency and local, but not overall, RNA thermodynamic stability. Mutations that modestly increase the local stability of the frameshift site RNA stem-loop structure increase frameshift efficiency 2-fold to 3-fold in cells. Thus, frameshift efficiency is determined by the strength of the thermodynamic barrier encountered by the ribosome. These data agree with previous in vitro measurements, suggesting that there are no virus- or host-specific factors that modulate frameshifting. The data also indicate that there are no sequence-specific requirements for the frameshift site stem-loop. A linear correlation between Gag-polymerase (Gag-Pol) levels in cells and levels in virions supports the idea of a stochastic virion assembly mechanism. We further demonstrate that the surrounding genomic RNA secondary structure influences frameshift efficiency and that a mutation that commonly arises in response to protease inhibitor therapy creates a functional but inefficient secondary slippery site. Finally, HIV-1 mutants with enhanced frameshift efficiencies are significantly less infectious, suggesting that compounds that increase frameshift efficiency by as little as 2-fold may be effective at suppressing HIV-1 replication. HIV, like many retroviruses, utilizes a -1 programmed ribosomal frameshift to generate viral enzymes in the form of a Gag-Pol polyprotein precursor. Thus, frameshifting is essential for viral replication. Here, we utilized a panel of mutant HIV strains to demonstrate that in cells, frameshifting efficiency is correlated with the stability of the local thermodynamic barrier to ribosomal translocation. Increasing the stability of the frameshift site RNA increases the frameshift efficiency 2-fold to 3-fold. Mutant viruses with increased frameshift efficiencies have significantly reduced infectivity. These data suggest that this effect might be exploited in the development of novel antiviral strategies.

Programmed −1 Frameshift of a Ribosome: Non-Monotonic Variation of Frameshift Efficiency with Increasing Stiffness of mRNA Secondary Structure

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).

Characterization of Ribosomal Frameshifting in Theiler's Murine Encephalomyelitis Virus.

ABSTRACTTheiler's murine encephalomyelitis virus (TMEV) is a member of the genus Cardiovirus in the Picornaviridae, a family of positive-sense single-stranded RNA viruses. Previously, we demonstrated that in the related cardiovirus, Encephalomyocarditis virus, a programmed −1 ribosomal frameshift (−1 PRF) occurs at a conserved G_GUU_UUU sequence within the 2B-encoding region of the polyprotein open reading frame (ORF). Here we show that −1 PRF occurs at a similar site during translation of the TMEV genome. In addition, we demonstrate that a predicted 3′ RNA stem-loop structure at a noncanonical spacing downstream of the shift site is required for efficient frameshifting in TMEV and that frameshifting also requires virus infection. Mutating the G_GUU_UUU shift site to inhibit frameshifting results in an attenuated virus with reduced growth kinetics and a small-plaque phenotype. Frameshifting in the virus context was found to be extremely efficient at 74 to 82%, which, to our knowledge, is the highest frameshifting efficiency recorded to date for any virus. We propose that highly efficient −1 PRF in TMEV provides a mechanism to escape the confines of equimolar expression normally inherent in the single-polyprotein expression strategy of picornaviruses.IMPORTANCE Many viruses utilize programmed −1 ribosomal frameshifting (−1 PRF) to produce different protein products at a defined ratio, or to translate overlapping ORFs to increase coding capacity. With few exceptions, −1 PRF occurs on specific “slippery” heptanucleotide sequences and is stimulated by RNA structure beginning 5 to 9 nucleotides (nt) downstream of the slippery site. Here we describe an unusual case of −1 PRF in Theiler's murine encephalomyelitis virus (TMEV) that is extraordinarily efficient (74 to 82% of ribosomes shift into the alternative reading frame) and, in stark contrast to other examples of −1 PRF, is dependent upon a stem-loop structure beginning 14 nt downstream of the slippery site. Furthermore, in TMEV-based reporter constructs in transfected cells, efficient frameshifting is critically dependent upon virus infection. We suggest that TMEV evolved frameshifting as a novel mechanism for removing ribosomes from the message (a “ribosome sink”) to downregulate synthesis of the 3′-encoded replication proteins.

Structure and Function of the HTLV‐1 pro‐pol Frameshift Site

Human T‐cell leukemia virus type I (HTLV‐1) is a human retrovirus. Replication of retroviruses, such as HTLV, is dependent upon synthesis of viral structural and enzymatic proteins. Translation of HTLV's enzymatic proteins depends upon two ‐1 programmed ribosomal frameshifts. While ‐1 programmed ribosomal frameshifting and the slippery sequences of the HTLV‐1 gag‐pro and pro‐pol frameshift sites have been established, the frameshift efficiencies and the structures within these sites have not been determined. Each frameshift site includes a heptanucleotide slippery sequence followed by a downstream RNA structure. In HTLV‐1's pro‐pol frameshift site, a pseudoknot structure is predicted downstream of the UUUAAAC slippery sequence. We hypothesize that this pseudoknot structure contributes significantly to frameshift efficiency. To test this hypothesis, we designed three variant frameshift sites, each of which disrupts a helix important to the pseudoknot structure. We report the in vitro frameshift efficiencies for the wild‐type and three variant HTLV pro‐pol frameshift sites. These efficiencies were measured using an in vitro dual‐luciferase frameshift assay. Preliminary data suggest that the pseudoknot structure is important to frameshifting.

Determination of the HTLV‐1 pro‐pol Frameshift Site Secondary Structure

Expression of human t‐cell leukemia virus type I (HTLV‐I) enzymes requires two ‐1 programmed ribosomal frameshifts. 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 ‐1 PRF and the slippery sequences of these frameshift sites have been established in HTLV‐I, their frameshift efficiencies and the structures within these sites have not been determined. In pro‐pol frameshift site, an RNA pseudoknot is predicted to fold downstream of the UUUAAAC slippery sequence. However, no structural data exist 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 and thermodynamics‐based secondary structure predictions are consistent with a pseudoknot secondary structure. Additionally, non‐denaturing PAGE analysis of the wild‐type structure and two mutant RNAs confirmed that the fold of the wild‐type RNA is significantly different from RNAs of identical length that cannot form a pseudoknot structure. These results establish the existence of a pseudoknot structure in the HTLV‐1 pro‐pol frameshift site.

The highly conserved codon following the slippery sequence supports -1 frameshift efficiency at the HIV-1 frameshift site.

HIV-1 utilises −1 programmed ribosomal frameshifting to translate structural and enzymatic domains in a defined proportion required for replication. A slippery sequence, U UUU UUA, and a stem-loop are well-defined RNA features modulating −1 frameshifting in HIV-1. The GGG glycine codon immediately following the slippery sequence (the ‘intercodon’) contributes structurally to the start of the stem-loop but has no defined role in current models of the frameshift mechanism, as slippage is inferred to occur before the intercodon has reached the ribosomal decoding site. This GGG codon is highly conserved in natural isolates of HIV. When the natural intercodon was replaced with a stop codon two different decoding molecules—eRF1 protein or a cognate suppressor tRNA—were able to access and decode the intercodon prior to −1 frameshifting. This implies significant slippage occurs when the intercodon is in the (perhaps distorted) ribosomal A site. We accommodate the influence of the intercodon in a model of frame maintenance versus frameshifting in HIV-1.

Monitoring ribosomal frameshifting as a platform to screen anti-riboswitch drug candidates.

Riboswitches are regions within mRNAs that can regulate downstream expression of genes through metabolite-induced alteration of their secondary structures. Due to the significant association of bacterial essential or virulence genes, bacterial riboswitches have become promising targets for development of putative antibacterial drugs. However, most of the screening systems to date are based on in vitro or bacterial systems, lacking the possibility to preobserve the adverse effects to the host's translation machinery. This chapter describes a novel screening method based on monitoring the riboswitch-induced -1 ribosomal frameshifting (-1 FS) efficiency in a mammalian cell-free lysate system using preQ1 class-I (preQ1-I) riboswitches as model target.

A stochastic model of translation with −1 programmed ribosomal frameshifting

Many viruses produce multiple proteins from a single mRNA sequence by encoding overlapping genes. One mechanism to decode both genes, which reside in alternate reading frames, is −1 programmed ribosomal frameshifting. Although recognized for over 25 years, the molecular and physical mechanism of −1 frameshifting remains poorly understood. We have developed a mathematical model that treats mRNA translation and associated −1 frameshifting as a stochastic process in which the transition probabilities are based on the energetics of local molecular interactions. The model predicts both the location and efficiency of −1 frameshift events in HIV-1. Moreover, we compute −1 frameshift efficiencies upon mutations in the viral mRNA sequence and variations in relative tRNA abundances, predictions that are directly testable in experiment.

High-Affinity Recognition of HIV-1 Frameshift-Stimulating RNA Alters Frameshifting in Vitro and Interferes with HIV-1 Infectivity

The life cycle of the human immunodeficiency virus type 1 (HIV-1) has an absolute requirement for ribosomal frameshifting during protein translation in order to produce the polyprotein precursor of the viral enzymes. While an RNA stem-loop structure (the “HIV-1 Frameshift Stimulating Signal”, or HIV-1 FSS) controls the frameshift efficiency and has been hypothesized as an attractive therapeutic target, developing compounds that selectively bind this RNA and interfere with HIV-1 replication has proven challenging. Building on our prior discovery of a “hit” molecule able to bind this stem-loop, we now report the development of compounds displaying high affinity for the HIV-1 FSS. These compounds are able to enhance frameshifting more than 50% in a dual-luciferase assay in human embryonic kidney cells, and they strongly inhibit the infectivity of pseudotyped HIV-1 virions.

Study of Polyribosomal Effects on Frameshifting

Frameshifting (FS) is a mechanism that enables the cell to use the same gene transcript to translate into different protein products. Frameshifting has been found mostly in prokaryotes and viruses. In prokaryotes, the mRNA for frameshifting usually contains three elements: a secondary structure (e.g., a hairpin), a slippery sequence and a Shine-Dalgarno (SD) sequence. When the ribosome is decoding the slippery sequence and stalled by the downstream structure and the upstream SD sequence, translocation of the ribosome may be affected and it will move backward by one base, resulting in −1 frameshifting.In our research, we use the FS motif from the E. coli dnaX gene as the model. We include three tandem repeats of the FS motif. It has been shown that the FS efficiency is about 80%. However, our experiments show that the efficiency was only about 30% at the first FS site, and about 80% at second and third FS sites. We speculate that polyribosomes may play a role for the results; when one ribosome still occupies the hairpin sequence and prevents its refolding, the following ribosome will encounter no structures and thus the FS efficiency is decreased. With a mutated sequence that effectively reduces the density of ribosomes on the mRNA, the FS efficiency of the first site was recovered back to 80%. Other experiments that change the extent of polyribosomes are ongoing to further explore the effects of polyribosomes on frameshifting.

Deciphering Ribosomal Frameshifting Dynamics

Ribosomes programmed by specific messenger RNA (mRNA) sequence elements can switch translation reading frames and synthesize different polypeptides from a single template. The Escherichia coli dnaX mRNA encodes two DNA polymerase III subunits, τ and γ, synthesized from 0-frame and probabilistic −1-slip across the slippery sequence: AAAAAAG. When further enhanced by structural barriers situated around the slippery sequence-an internal Shine-Dalgarno sequence and a stable hairpin stem loop, an 80% (= γ/(γ+τ)) frameshift efficiency is attained. Here, we attempt to determine the frameshift timing within one translation cycle by following a single ribosome translating a frameshift-promoting mRNA held on optical tweezers. In parallel, by mass spectrometry (MS), we survey the synthesized polypeptides to resolve where on the mRNA the ribosome has slipped.From the mass spectra of polypeptides terminated at the −1-frame stop codon, we learned that the ribosome −1-slips from more than one codon position around the slippery sequence. Some −1-frameshifted polypeptides were found to bear an extra amino acid, or to lack one, indicating that slipping sizes of −4 and +2-nt also occurred. Similarly, distinctive large-scale fluctuating translocation dynamics were seen in our real-time single-ribosome translation trajectories. This reveals that a translocating ribosome can explore a broad range of frameshift pathways. Frequently adopted frameshift pathways, i.e. the more abundant frameshifted species resolved by MS, exhibit a preference for minimizing codon:anticodon base-pair mismatches on the ribosome after a slip. Mismatch-containing ribosomes can be prematurely terminated by release factors, resulting in release of incomplete peptides. Indeed, we observed higher yields of incomplete peptides that are terminated at frameshift sites where significant mismatches were encountered. These species coincide with the prematurely stalled ribosomes recorded in the translation trajectories. Collectively what emerges from our results is a versatile ribosomal frameshifting scheme during mRNA translocation, facilitating branching of frameshift pathways.

An mRNA Downstream Structure Promotes Frameshifting by Destabilizing the Hybrid State and Impeding the EF-G Driven Translocation Process

Translational frameshifting occurs when a ribosome slips one or two nucleotides on a messenger RNA and generates a new sequence of amino acids. Many viral RNAs have programed frameshift-promoting signals to produce their proteins in the precise ratio needed for their viability. We used single-molecule fluorescence resonance energy transfer (smFRET) to study the dynamics of −1 programmed frameshifting by the dnaX gene in E. coli. The frameshifting mRNA has the usual three prokaryotic frameshifting signals: an internal Shine-Dalgarno sequence, a slippery sequence, and a stem loop. One round of translational elongation of the slippery sequence was characterized by the FRET changes between a Cy3-labeled L1 stalk in the 50S subunit and a Cy5-tRNALys in the P-site. We observed that the downstream stem loop, a critical signal for efficient frameshifting, destabilizes the hybrid state and thus shifts the equilibrium toward to the classical state of pre-translocation complexes. Translocation catalyzed by EF-G was significantly slower in the frameshifting mRNA than in the non-frameshifting mRNA lacking the stem loop. Furthermore, pre-translocation complexes of the frameshifting mRNA underwent several transitions between the classical and hybrid states in the presence of EF-G prior to complete translocation, while the majority of the non-frameshifting mRNA translocated rapidly via a single hybrid state. Quantitative analysis showed that the stem loop impedes EF-G driven translocation in the 30S subunit by elevating the activation barriers to translocation, and leaves the EF-G bound-hybrid state in dynamic equilibrium with the hybrid and classical states. We propose that by keeping the ribosome and tRNAs in the dynamically transiting pre-translocation states, the frameshifting mRNA allows more time for the ribosome to explore other paths, such as −1 frameshifting.

Unusual −1 Ribosomal Frameshift Caused by Stable RNA G-Quadruplex in Open Reading Frame

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.

Altering SARS Coronavirus Frameshift Efficiency Affects Genomic and Subgenomic RNA Production

In previous studies, differences in the amount of genomic and subgenomic RNA produced by coronaviruses with mutations in the programmed ribosomal frameshift signal of ORF1a/b were observed. It was not clear if these differences were due to changes in genomic sequence, the protein sequence or the frequency of frameshifting. Here, viruses with synonymous codon changes are shown to produce different ratios of genomic and subgenomic RNA. These findings demonstrate that the protein sequence is not the primary cause of altered genomic and subgenomic RNA production. The synonymous codon changes affect both the structure of the frameshift signal and frameshifting efficiency. Small differences in frameshifting efficiency result in dramatic differences in genomic RNA production and TCID50 suggesting that the frameshifting frequency must stay above a certain threshold for optimal virus production. The data suggest that either the RNA sequence or the ratio of viral proteins resulting from different levels of frameshifting affects viral replication.

Altering SARS Coronavirus Frameshift Efficiency Affects Genomic and Subgenomic RNA Production

Altering SARS Coronavirus Frameshift Efficiency Affects Genomic and Subgenomic RNA Production