Hybrid Gene Origination Creates Human-Virus Chimeric Proteins during Infection.

RNA viruses are a major human health threat. The life cycles of many highly pathogenic RNA viruses like influenza A virus (IAV) and Lassa virus depends on host mRNA, because viral polymerases cleave 5'-m7G-capped host transcripts to prime viral mRNA synthesis ("cap-snatching"). We hypothesized that start codons within cap-snatched host transcripts could generate chimeric human-viral mRNAs with coding potential. We report the existence of this mechanism of gene origination, which we named "start-snatching." Depending on the reading frame, start-snatching allows the translation of host and viral "untranslated regions" (UTRs) to create N-terminally extended viral proteins or entirely novel polypeptides by genetic overprinting. We show that both types of chimeric proteins are made in IAV-infected cells, generate T cell responses, and contribute to virulence. Our results indicate that during infection with IAV, and likely a multitude of other human, animal and plant viruses, a host-dependent mechanism allows the genesis of hybrid genes.

Manipulation of the unfolded protein response: A pharmacological strategy against coronavirus infection.

Coronavirus infection induces the unfolded protein response (UPR), a cellular signalling pathway composed of three branches, triggered by unfolded proteins in the endoplasmic reticulum (ER) due to high ER load. We have used RNA sequencing and ribosome profiling to investigate holistically the transcriptional and translational response to cellular infection by murine hepatitis virus (MHV), often used as a model for the Betacoronavirus genus to which the recently emerged SARS-CoV-2 also belongs. We found the UPR to be amongst the most significantly up-regulated pathways in response to MHV infection. To confirm and extend these observations, we show experimentally the induction of all three branches of the UPR in both MHV- and SARS-CoV-2-infected cells. Over-expression of the SARS-CoV-2 ORF8 or S proteins alone is itself sufficient to induce the UPR. Remarkably, pharmacological inhibition of the UPR greatly reduced the replication of both MHV and SARS-CoV-2, revealing the importance of this pathway for successful coronavirus replication. This was particularly striking when both IRE1α and ATF6 branches of the UPR were inhibited, reducing SARS-CoV-2 virion release (~1,000-fold). Together, these data highlight the UPR as a promising antiviral target to combat coronavirus infection.

Investigating molecular mechanisms of 2A-stimulated ribosomal pausing and frameshifting in Theilovirus.

The 2A protein of Theiler's murine encephalomyelitis virus (TMEV) acts as a switch to stimulate programmed -1 ribosomal frameshifting (PRF) during infection. Here, we present the X-ray crystal structure of TMEV 2A and define how it recognises the stimulatory RNA element. We demonstrate a critical role for bases upstream of the originally predicted stem-loop, providing evidence for a pseudoknot-like conformation and suggesting that the recognition of this pseudoknot by beta-shell proteins is a conserved feature in cardioviruses. Through examination of PRF in TMEV-infected cells by ribosome profiling, we identify a series of ribosomal pauses around the site of PRF induced by the 2A-pseudoknot complex. Careful normalisation of ribosomal profiling data with a 2A knockout virus facilitated the identification, through disome analysis, of ribosome stacking at the TMEV frameshifting signal. These experiments provide unparalleled detail of the molecular mechanisms underpinning Theilovirus protein-stimulated frameshifting.

Macrolide-induced ribosomal frameshifting: a new route to antibiotic resistance.

In this issue of Molecular Cell, Gupta et al. (2013a) describe a novel, antibiotic-dependent ribosomal frameshifting event that activates translation of an antibiotic resistance gene.

Characterization of the stimulators of protein-directed ribosomal frameshifting in Theiler's murine encephalomyelitis virus.

Many viruses utilize programmed -1 ribosomal frameshifting (-1 PRF) to express additional proteins or to produce frameshift and non-frameshift protein products at a fixed stoichiometric ratio. PRF is also utilized in the expression of a small number of cellular genes. Frameshifting is typically stimulated by signals contained within the mRNA: a 'slippery' sequence and a 3'-adjacent RNA structure. Recently, we showed that -1 PRF in encephalomyocarditis virus (EMCV) is trans-activated by the viral 2A protein, leading to a temporal change in PRF efficiency from 0% to 70% during virus infection. Here we analyzed PRF in the related Theiler's murine encephalomyelitis virus (TMEV). We show that 2A is also required for PRF in TMEV and can stimulate PRF to levels as high as 58% in rabbit reticulocyte cell-free translations and 81% during virus infection. We also show that TMEV 2A trans-activates PRF on the EMCV signal but not vice versa. We present an extensive mutational analysis of the frameshift stimulators (mRNA signals and 2A protein) analysing activity in in vitro translation, electrophoretic mobility shift and in vitro ribosome pausing assays. We also investigate the PRF mRNA signal with RNA structure probing. Our results substantially extend previous characterization of protein-stimulated PRF.

Comparative Analysis of Gene Expression in Virulent and Attenuated Strains of Infectious Bronchitis Virus at Subcodon Resolution.

Like all coronaviruses, avian infectious bronchitis virus (IBV) possesses a long, single-stranded, positive-sense RNA genome (∼27 kb) and has a complex replication strategy that includes the production of a nested set of subgenomic mRNAs (sgmRNAs). Here, we used whole-transcriptome sequencing (RNASeq) and ribosome profiling (RiboSeq) to delineate gene expression in the IBV M41-CK and Beau-R strains at subcodon resolution. RNASeq facilitated a comparative analysis of viral RNA synthesis and revealed two novel transcription junction sites in the attenuated Beau-R strain, one of which would generate a sgmRNA encoding a ribosomally occupied open reading frame (dORF) located downstream of the nucleocapsid coding region. RiboSeq permitted quantification of the translational efficiency of virus gene expression and identified, for the first time, sites of ribosomal pausing on the genome. Quantification of reads flanking the programmed ribosomal frameshifting (PRF) signal at the genomic RNA ORF1a/ORF1b junction revealed that PRF in IBV is highly efficient (33 to 40%). Triplet phasing of RiboSeq data allowed precise determination of reading frames and revealed the translation of two ORFs (ORF4b and ORF4c on sgmRNA IR), which are widely conserved across IBV isolates. Analysis of differential gene expression in infected primary chick kidney cells indicated that the host cell response to IBV occurs primarily at the level of transcription, with global upregulation of immune-related mRNA transcripts following infection and comparatively modest changes in the translation efficiencies of host genes. Cellular genes and gene networks differentially expressed during virus infection were also identified, giving insights into the host cell response to IBV infection.IMPORTANCE IBV is a major avian pathogen and presents a substantial economic burden to the poultry industry. Improved vaccination strategies are urgently needed to curb the global spread of this virus, and the development of suitable vaccine candidates will be aided by an improved understanding of IBV molecular biology. Our high-resolution data have enabled a precise study of transcription and translation in cells infected with both pathogenic and attenuated forms of IBV and expand our understanding of gammacoronaviral gene expression. We demonstrate that gene expression shows considerable intraspecies variation, with single nucleotide polymorphisms being associated with altered production of sgmRNA transcripts, and our RiboSeq data sets enabled us to uncover novel ribosomally occupied ORFs in both strains. The numerous cellular genes and gene networks found to be differentially expressed during virus infection provide insights into the host cell response to IBV infection.

Programmed -2/-1 Ribosomal Frameshifting in Simarteriviruses: an Evolutionarily Conserved Mechanism.

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, nsp1ß, 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 nsp1ß 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 nsp1ß 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 nsp1ß, 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 nsp1ßs 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.

Ribosome profiling of the retrovirus murine leukemia virus.

BACKGROUND: The retrovirus murine leukemia virus (MuLV) has an 8.3 kb RNA genome with a simple 5'-gag-pol-env-3' architecture. Translation of the pol gene is dependent upon readthrough of the gag UAG stop codon; whereas the env gene is translated from spliced mRNA transcripts. Here, we report the first high resolution analysis of retrovirus gene expression through tandem ribosome profiling (RiboSeq) and RNA sequencing (RNASeq) of MuLV-infected cells. RESULTS: Ribosome profiling of MuLV-infected cells was performed, using the translational inhibitors harringtonine and cycloheximide to distinguish initiating and elongating ribosomes, respectively. Meta-analyses of host cell gene expression demonstrated that the RiboSeq datasets specifically captured the footprints of translating ribosomes at high resolution. Direct measurement of ribosomal occupancy of the MuLV genomic RNA indicated that ~ 7% of ribosomes undergo gag stop codon readthrough to access the pol gene. Initiation of translation was found to occur at several additional sites within the 5' leaders of the gag and env transcripts, upstream of their respective annotated start codons. CONCLUSIONS: These experiments reveal the existence of a number of previously uncharacterised, ribosomally occupied open reading frames within the MuLV genome, with possible regulatory consequences. In addition, we provide the first direct measurements of stop codon readthrough efficiency during cellular infection.

High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling.

Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global "snap-shot" of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal frameshift site. To our knowledge this is the first application of ribosome profiling to an RNA virus.

A novel role for poly(C) binding proteins in programmed ribosomal frameshifting.

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 nsp1ß. Here we show that the the trans-activation of frameshifting is carried out by a protein complex composed of nsp1ß 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/nsp1ß 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.

The use of duplex-specific nuclease in ribosome profiling and a user-friendly software package for Ribo-seq data analysis.

Ribosome profiling is a technique that permits genome-wide, quantitative analysis of translation and has found broad application in recent years. Here we describe a modified profiling protocol and software package designed to benefit more broadly the translation community in terms of simplicity and utility. The protocol, applicable to diverse organisms, including organelles, is based largely on previously published profiling methodologies, but uses duplex-specific nuclease (DSN) as a convenient, species-independent way to reduce rRNA contamination. We show that DSN-based depletion compares favorably with other commonly used rRNA depletion strategies and introduces little bias. The profiling protocol typically produces high levels of triplet periodicity, facilitating the detection of coding sequences, including upstream, downstream, and overlapping open reading frames (ORFs) and an alternative ribosome conformation evident during termination of protein synthesis. In addition, we provide a software package that presents a set of methods for parsing ribosomal profiling data from multiple samples, aligning reads to coding sequences, inferring alternative ORFs, and plotting average and transcript-specific aspects of the data. Methods are also provided for extracting the data in a form suitable for differential analysis of translation and translational efficiency.

Inhibition of Translation Initiation by Protein 169: A Vaccinia Virus Strategy to Suppress Innate and Adaptive Immunity and Alter Virus Virulence.

Vaccinia virus (VACV) is the prototypic orthopoxvirus and the vaccine used to eradicate smallpox. Here we show that VACV strain Western Reserve protein 169 is a cytoplasmic polypeptide expressed early during infection that is excluded from virus factories and inhibits the initiation of cap-dependent and cap-independent translation. Ectopic expression of protein 169 causes the accumulation of 80S ribosomes, a reduction of polysomes, and inhibition of protein expression deriving from activation of multiple innate immune signaling pathways. A virus lacking 169 (vΔ169) replicates and spreads normally in cell culture but is more virulent than parental and revertant control viruses in intranasal and intradermal murine models of infection. Intranasal infection by vΔ169 caused increased pro-inflammatory cytokines and chemokines, infiltration of pulmonary leukocytes, and lung weight. These alterations in innate immunity resulted in a stronger CD8+ T-cell memory response and better protection against virus challenge. This work illustrates how inhibition of host protein synthesis can be a strategy for virus suppression of innate and adaptive immunity.

Transactivation of programmed ribosomal frameshifting by a viral protein.

Programmed -1 ribosomal frameshifting (-1 PRF) is a widely used translational mechanism facilitating the expression of two polypeptides from a single mRNA. Commonly, the ribosome interacts with an mRNA secondary structure that promotes -1 frameshifting on a homopolymeric slippery sequence. Recently, we described an unusual -2 frameshifting (-2 PRF) signal directing efficient expression of a transframe protein [nonstructural protein 2TF (nsp2TF)] of porcine reproductive and respiratory syndrome virus (PRRSV) from an alternative reading frame overlapping the viral replicase gene. Unusually, this arterivirus PRF signal lacks an obvious stimulatory RNA secondary structure, but as confirmed here, can also direct the occurrence of -1 PRF, yielding a third, truncated nsp2 variant named "nsp2N." Remarkably, we now show that both -2 and -1 PRF are transactivated by a protein factor, specifically a PRRSV replicase subunit (nsp1ß). Embedded in nsp1ß's papain-like autoproteinase domain, we identified a highly conserved, putative RNA-binding motif that is critical for PRF transactivation. The minimal RNA sequence required for PRF was mapped within a 34-nt region that includes the slippery sequence and a downstream conserved CCCANCUCC motif. Interaction of nsp1ß with the PRF signal was demonstrated in pull-down assays. These studies demonstrate for the first time, to our knowledge, that a protein can function as a transactivator of ribosomal frameshifting. The newly identified frameshifting determinants provide potential antiviral targets for arterivirus disease control and prevention. Moreover, protein-induced transactivation of frameshifting may be a widely used mechanism, potentially including previously undiscovered viral strategies to regulate viral gene expression and/or modulate host cell translation upon infection.

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

Theiler'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.

Modulation of stop codon read-through efficiency and its effect on the replication of murine leukemia virus.

UNLABELLED: Translational readthrough--suppression of termination at a stop codon--is exploited in the replication cycles of several viruses and represents a potential target for antiviral intervention. In the gammaretroviruses, typified by Moloney murine leukemia virus (MuLV), gag and pol are in the same reading frame, separated by a UAG stop codon, and termination codon readthrough is required for expression of the viral Gag-Pol fusion protein. Here, we investigated the effect on MuLV replication of modulating readthrough efficiency. We began by manipulating the readthrough signal in the context of an infectious viral clone to generate a series of MuLV variants in which readthrough was stimulated or reduced. In carefully controlled infectivity assays, it was found that reducing the MuLV readthrough efficiency only 4-fold led to a marked defect and that a 10-fold reduction essentially abolished replication. However, up to an ∼ 8.5-fold stimulation of readthrough (up to 60% readthrough) was well tolerated by the virus. These high levels of readthrough were achieved using a two-plasmid system, with Gag and Gag-Pol expressed from separate infectious clones. We also modulated readthrough by silencing expression of eukaryotic release factors 1 and 3 (eRF1 and eRF3) or by introducing aminoglycosides into the cells. The data obtained indicate that gammaretroviruses tolerate a substantial excess of viral Gag-Pol synthesis but are very sensitive to a reduction in levels of this polyprotein. Thus, as is also the case for ribosomal frameshifting, antiviral therapies targeting readthrough with inhibitory agents are likely to be the most beneficial. IMPORTANCE: Many pathogenic RNA viruses and retroviruses use ribosomal frameshifting or stop codon readthrough to regulate expression of their replicase enzymes. These translational "recoding" processes are potential targets for antiviral intervention, but we have only a limited understanding of the consequences to virus replication of modulating the efficiency of recoding, particularly for those viruses employing readthrough. In this paper, we describe the first systematic analysis of the effect of increasing or decreasing readthrough efficiency on virus replication using the gammaretrovirus MuLV as a model system. We find unexpectedly that MuLV replication is only slightly inhibited by substantial increases in readthrough frequency, but as with other viruses that use recoding strategies, replication is quite sensitive to even modest reductions. These studies provide insights into both the readthrough process and MuLV replication and have implications for the selection of antivirals against gammaretroviruses.

Identification of immune and viral correlates of norovirus protective immunity through comparative study of intra-cluster norovirus strains.

Whether or not primary norovirus infections induce protective immunity has become a controversial issue, potentially confounded by the comparison of data from genetically distinct norovirus strains. Early human volunteer studies performed with a norovirus-positive inoculum initially led to the conclusion that primary infection does not generate long-term, protective immunity. More recently though, the epidemiological pattern of norovirus pandemics has led to the extrapolation that primary norovirus infection induces herd immunity. While these are seemingly discordant observations, they may in fact reflect virus strain-, cluster-, or genogroup-specific differences in protective immunity induction. Here, we report that highly genetically related intra-cluster murine norovirus strains differ dramatically in their ability to induce a protective immune response: Primary MNV-3 infection induced robust and cross-reactive protection, whereas primary MNV-1 infection induced modest homotypic and no heterotypic protection. In addition to this fundamental observation that intra-cluster norovirus strains display remarkable differences in protective immunity induction, we report three additional important observations relevant to norovirus:host interactions. First, antibody and CD4⁺ T cells are essential to controlling secondary norovirus infections. Second, the viral minor structural protein VP2 regulates the maturation of antigen presenting cells and protective immunity induction in a virus strain-specific manner, pointing to a mechanism by which MNV-1 may prevent the stimulation of memory immune responses. Third, VF1-mediated regulation of cytokine induction also correlates with protective immunity induction. Thus, two highly genetically-related norovirus strains displayed striking differences in induction of protective immune responses, strongly suggesting that the interpretation of norovirus immunity and vaccine studies must consider potential virus strain-specific effects. Moreover, we have identified immune (antibody and CD4⁺ T cells) and viral (VP2 and possibly VF1) correlates of norovirus protective immunity. These findings have significant implications for our understanding of norovirus immunity during primary infections as well as the development of new norovirus vaccines.

Characterization of the stop codon readthrough signal of Colorado tick fever virus segment 9 RNA.

Termination codon readthrough is utilized as a mechanism of expression of a growing number of viral and cellular proteins, but in many cases the mRNA signals that promote readthrough are poorly characterized. Here, we investigated the readthrough signal of Colorado tick fever virus (CTFV) segment 9 RNA (Seg-9). CTFV is the type-species of the genus Coltivirus within the family Reoviridae and is a tick-borne, double-stranded, segmented RNA virus. Seg-9 encodes a 36-kDa protein VP9, and by readthrough of a UGA stop codon, a 65-kDa product, VP9'. Using a reporter system, we defined the minimal sequence requirements for readthrough and confirmed activity in both mammalian and insect cell-free translation systems, and in transfected mammalian cells. Mutational analysis revealed that readthrough was UGA specific, and that the local sequence context around the UGA influenced readthrough efficiency. Readthrough was also dependent upon a stable RNA stem-loop structure beginning eight bases downstream from the UGA codon. Mutational analysis of this stem-loop revealed a requirement for the stem region but not for substructures identified within the loop. Unexpectedly, we were unable to detect a ribosomal pause during translation of the CTFV signal, suggesting that the mechanism of readthrough, at least at this site, is unlikely to be dependent upon RNA secondary-structure induced ribosomal pausing at the recoded stop codon.

Spacer-length dependence of programmed -1 or -2 ribosomal frameshifting on a U6A heptamer supports a role for messenger RNA (mRNA) tension in frameshifting.

Programmed -1 ribosomal frameshifting is employed in the expression of a number of viral and cellular genes. In this process, the ribosome slips backwards by a single nucleotide and continues translation of an overlapping reading frame, generating a fusion protein. Frameshifting signals comprise a heptanucleotide slippery sequence, where the ribosome changes frame, and a stimulatory RNA structure, a stem-loop or RNA pseudoknot. Antisense oligonucleotides annealed appropriately 3' of a slippery sequence have also shown activity in frameshifting, at least in vitro. Here we examined frameshifting at the U6A slippery sequence of the HIV gag/pol signal and found high levels of both -1 and -2 frameshifting with stem-loop, pseudoknot or antisense oligonucleotide stimulators. By examining -1 and -2 frameshifting outcomes on mRNAs with varying slippery sequence-stimulatory RNA spacing distances, we found that -2 frameshifting was optimal at a spacer length 1-2 nucleotides shorter than that optimal for -1 frameshifting with all stimulatory RNAs tested. We propose that the shorter spacer increases the tension on the mRNA such that when the tRNA detaches, it more readily enters the -2 frame on the U6A heptamer. We propose that mRNA tension is central to frameshifting, whether promoted by stem-loop, pseudoknot or antisense oligonucleotide stimulator.

Modulation of ribosomal frameshifting frequency and its effect on the replication of Rous sarcoma virus.

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.

Structural and Functional Insights into Viral Programmed Ribosomal Frameshifting.

Protein synthesis by the ribosome is the final stage of biological information transfer and represents an irreversible commitment to gene expression. Accurate translation of messenger RNA is therefore essential to all life, and spontaneous errors by the translational machinery are highly infrequent (∼1/100,000 codons). Programmed -1 ribosomal frameshifting (-1PRF) is a mechanism in which the elongating ribosome is induced at high frequency to slip backward by one nucleotide at a defined position and to continue translation in the new reading frame. This is exploited as a translational regulation strategy by hundreds of RNA viruses, which rely on -1PRF during genome translation to control the stoichiometry of viral proteins. While early investigations of -1PRF focused on virological and biochemical aspects, the application of X-ray crystallography and cryo-electron microscopy (cryo-EM), and the advent of deep sequencing and single-molecule approaches have revealed unexpected structural diversity and mechanistic complexity. Molecular players from several model systems have now been characterized in detail, both in isolation and, more recently, in the context of the elongating ribosome. Here we provide a summary of recent advances and discuss to what extent a general model for -1PRF remains a useful way of thinking.