The human immunodeficiency virus type 1 ribosomal frameshifting site is an invariant sequence determinant and an important target for antiviral therapy

Human immunodeficiency virus type 1 (HIV-1) utilizes a distinctive form of gene regulation as part of its life cycle, termed programmed -1 ribosomal frameshifting, to produce the required ratio of the Gag and Gag-Pol polyproteins. We carried out a sequence comparison of 1,000 HIV-1 sequences at the slippery site (UUUUUUA) and found that the site is invariant, which is somewhat surprising for a virus known for its variability. This prompted us to prepare a series of mutations to examine their effect upon frameshifting and viral infectivity. Among the series of mutations were changes of the HIV-1 slippery site to those effectively utilized by other viruses, because such mutations would be anticipated to have a relatively mild effect upon frameshifting. The results demonstrate that any change to the slippery site reduced frameshifting levels and also dramatically inhibited infectivity. Because ribosomal frameshifting is essential for HIV-1 replication and it is surprisingly resistant to mutation, modulation of HIV-1 frameshifting efficiency potentially represents an important target for the development of novel antiviral therapeutics.     

Ribosomal pausing during translation of an RNA pseudoknot.

The genomic RNA of the coronavirus infectious bronchitis virus contains an efficient ribosomal frameshift signal which comprises a heptanucleotide slippery sequence followed by an RNA pseudoknot structure. The presence of the pseudoknot is essential for high-efficiency frameshifting, and it has been suggested that its function may be to slow or stall the ribosome in the vicinity of the slippery sequence. To test this possibility, we have studied translational elongation in vitro on mRNAs engineered to contain a well-defined pseudoknot-forming sequence. Insertion of the pseudoknot at a specific location within the influenza virus PB1 mRNA resulted in the production of a new translational intermediate corresponding to the size expected for ribosomal arrest at the pseudoknot. The appearance of this protein was transient, indicating that it was a true paused intermediate rather than a dead-end product, and mutational analysis confirmed that its appearance was dependent on the presence of a pseudoknot structure within the mRNA. These observations raise the possibility that a pause is required for the frameshift process. The extent of pausing at the pseudoknot was compared with that observed at a sequence designed to form a simple stem-loop structure with the same base pairs as the pseudoknot. This structure proved to be a less effective barrier to the elongating ribosome than the pseudoknot and in addition was unable to direct efficient ribosomal frameshifting, as would be expected if pausing plays an important role in frameshifting. However, the stem-loop was still able to induce significant pausing, and so this effect alone may be insufficient to account for the contribution of the pseudoknot to frameshifting.     

Ribosomal Pausing at a Frameshifter RNA Pseudoknot Is Sensitive to Reading Phase but Shows Little Correlation with Frameshift Efficiency

Here we investigated ribosomal pausing at sites of programmed -1 ribosomal frameshifting, using translational elongation and ribosome heelprint assays. The site of pausing at the frameshift signal of infectious bronchitis virus (IBV) was determined and was consistent with an RNA pseudoknot-induced pause that placed the ribosomal P- and A-sites over the slippery sequence. Similarly, pausing at the simian retrovirus 1 gag/pol signal, which contains a different kind of frameshifter pseudoknot, also placed the ribosome over the slippery sequence, supporting a role for pausing in frameshifting. However, a simple correlation between pausing and frameshifting was lacking. Firstly, a stem-loop structure closely related to the IBV pseudoknot, although unable to stimulate efficient frameshifting, paused ribosomes to a similar extent and at the same place on the mRNA as a parental pseudoknot. Secondly, an identical pausing pattern was induced by two pseudoknots differing only by a single loop 2 nucleotide yet with different functionalities in frameshifting. The final observation arose from an assessment of the impact of reading phase on pausing. Given that ribosomes advance in triplet fashion, we tested whether the reading frame in which ribosomes encounter an RNA structure (the reading phase) would influence pausing. We found that the reading phase did influence pausing but unexpectedly, the mRNA with the pseudoknot in the phase which gave the least pausing was found to promote frameshifting more efficiently than the other variants. Overall, these experiments support the view that pausing alone is insufficient to mediate frameshifting and additional events are required. The phase dependence of pausing may be indicative of an activity in the ribosome that requires an optimal contact with mRNA secondary structures for efficient unwinding.     

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 U(6)A 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 U(6)A heptamer. We propose that mRNA tension is central to frameshifting, whether promoted by stem-loop, pseudoknot or antisense oligonucleotide stimulator.     

Mutational analysis of the "slippery-sequence" component of a coronavirus ribosomal frameshifting signal.

The ribosomal frameshift signal in the genomic RNA of the coronavirus IBV is composed of two elements, a heptanucleotide "slippery-sequence" and a downstream RNA pseudoknot. We have investigated the kinds of slippery sequence that can function at the IBV frameshift site by analysing the frameshifting properties of a series of slippery-sequence mutants. We firstly confirmed that the site of frameshifting in IBV was at the heptanucleotide stretch UUUAAAC, and then used our knowledge of the pseudoknot structure and a suitable reporter gene to prepare an expression construct that allowed both the magnitude and direction of ribosomal frameshifting to be determined for candidate slippery sequences. Our results show that in almost all of the sequences tested, frameshifting is strictly into the -1 reading frame. Monotonous runs of nucleotides, however, gave detectable levels of a -2/+1 frameshift product, and U stretches in particular gave significant levels (2% to 21%). Preliminary evidence suggests that the RNA pseudoknot may play a role in influencing frameshift direction. The spectrum of slip-sequences tested in this analysis included all those known or suspected to be utilized in vivo. Our results indicate that triplets of A, C, G and U are functional when decoded in the ribosomal P-site following slippage (XXXYYYN) although C triplets were the least effective. In the A-site (XXYYYYN), triplets of C and G were non-functional. The identity of the nucleotide at position 7 of the slippery sequence (XXXYYYN) was found to be a critical determinant of frameshift efficiency and we show that a hierarchy of frameshifting exists for A-site codons. These observations lead us to suggest that ribosomal frameshifting at a particular site is determined, at least in part, by the strength of the interaction of normal cellular tRNAs with the A-site codon and does not necessarily involve specialized "shifty" tRNAs.     

Predicting ribosomal frameshifting efficiency

Many retroviruses use -1 ribosomal frameshifting as part of the mechanism in translational control of viral protein synthesis. Quantitative prediction of the efficiency of -1 frameshifting is crucial for understanding the viral gene expression. Here we investigate the free energy landscape for a minimal -1 programmed ribosomal frameshifting machinery, including the codon-anticodon base pairs at the slippery site, the downstream messenger RNA structure and the spacer between the slippery site and the downstream structure. The free energy landscape analysis leads to a quantitative relationship between the frameshifting efficiency and the tension force generated during the movement of codon-anticodon complexes, which may occur in the A/T to A/A accommodation process or the translocation process. The analysis shows no consistent correlation between frameshifting efficiency and global stability of the downstream mRNA structure.     

Comparative mutational analysis of cis-acting RNA signals for translational frameshifting in HIV-1 and HTLV-2

Human immunodeficiency virus type 1 (HIV-1) and human T cell leukemia virus type II (HTLV-2) use a similar mechanism for –1 translational frameshifting to overcome the termination codon in viral RNA at the end of the gag gene. Previous studies have identified two important RNA signals for frameshifting, the slippery sequence and a downstream stem–loop structure. However, there have been somewhat conflicting reports concerning the individual contributions of these sequences. In this study we have performed a comprehensive mutational analysis of the cis-acting RNA sequences involved in HIV-1 gag–pol and HTLV-2 gag–pro frameshifting. Using an in vitro translation system we determined frameshifting efficiencies for shuffled HIV-1/HTLV-2 RNA elements in a background of HIV-1 or HTLV-2 sequences. We show that the ability of the slippery sequence and stem–loop to promote ribosomal frameshifting is influenced by the flanking upstream sequence and the nucleotides in the spacer element. A wide range of frameshift efficiency rates was observed for both viruses when shuffling single sequence elements. The results for HIV-1/HTLV-2 chimeric constructs represent strong evidence supporting the notion that the viral wild-type sequences are not designed for maximal frameshifting activity but are optimized to a level suited to efficient viral replication.     

Programmed ribosomal frameshifting in SIV is induced by a highly structured RNA stem-loop

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.     

Ribosomal frameshifting requires a pseudoknot in the Saccharomyces cerevisiae double-stranded RNA virus.

The large double-stranded RNA of the Saccharomyces cerevisiae (yeast) virus has two large overlapping open reading frames on the plus strand, one of which is translated via a -1 ribosomal frameshift. Sequences including the overlapping region, placed in novel contexts, can direct ribosomes to make a -1 frameshift in wheat germ extract, Escherichia coli and S. cerevisiae. This sequence includes a consensus slippery sequence, GGGUUUA, and has the potential to form a pseudoknot 3' to the putative frameshift site. Based on deletion analysis, a region of 71 nucleotides including the potential pseudoknot and the putative slippery sequence is sufficient for frameshifting. Site-directed mutagenesis demonstrates that the pseudoknot is essential for frameshifting.     

Structural and functional studies of retroviral RNA pseudoknots involved in ribosomal frameshifting: nucleotides at the junction of the two stems are important for efficient ribosomal frameshifting.

Ribosomal frameshifting, a translational mechanism used during retroviral replication, involves a directed change in reading frame at a specific site at a defined frequency. Such programmed frameshifting at the mouse mammary tumor virus (MMTV) gag-pro shift site requires two mRNA signals: a heptanucleotide shifty sequence and a pseudoknot structure positioned downstream. Using in vitro translation assays and enzymatic and chemical probes for RNA structure, we have defined features of the pseudoknot that promote efficient frameshifting. Heterologous RNA structures, e.g. a hairpin, a tRNA or a synthetic pseudoknot, substituted downstream of the shifty site fail to promote frameshifting, suggesting that specific features of the MMTV pseudoknot are important for function. Site-directed mutations of the MMTV pseudoknot indicate that the pseudoknot junction, including an unpaired adenine nucleotide between the two stems, provides a specific structural determinant for efficient frameshifting. Pseudoknots derived from other retroviruses (i.e. the feline immunodeficiency virus and the simian retrovirus type 1) also promote frameshifting at the MMTV gag-pro shift site, dependent on the same structure at the junction of the two stems.     

Structure, stability and function of RNA pseudoknots involved in stimulating ribosomal frameshifting

Programmed -1 ribosomal frameshifting has become the subject of increasing interest over the last several years, due in part to the ubiquitous nature of this translational recoding mechanism in pathogenic animal and plant viruses. All cis-acting frameshift signals encoded in mRNAs are minimally composed of two functional elements: a heptanucleotide "slippery sequence" conforming to the general form X XXY YYZ, followed by an RNA structural element, usually an H-type RNA pseudoknot, positioned an optimal number of nucleotides (5 to 9) downstream. The slippery sequence itself promotes a low level ( approximately 1 %) of frameshifting; however, downstream pseudoknots stimulate this process significantly, in some cases up to 30 to 50 %. Although the precise molecular mechanism of stimulation of frameshifting remains poorly understood, significant advances have been made in our knowledge of the three-dimensional structures, thermodynamics of folding, and functional determinants of stimulatory RNA pseudoknots derived from the study of several well-characterized frameshift signals. These studies are summarized here and provide new insights into the structural requirements and mechanism of programmed -1 ribosomal frameshifting.     

Stimulation of ribosomal frameshifting by antisense LNA

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.     

Structural requirements for efficient translational frameshifting in the synthesis of the putative viral RNA-dependent RNA polymerase of potato leafroll virus.

The putative RNA-dependent RNA polymerase of potato leafroll luteovirus (PLRV) is expressed by -1 ribosomal frameshifting in the region where the open reading frames (ORF) of proteins 2a and 2b overlap. The signal responsible for efficient frameshift is composed of the slippery site UUUAAAU followed by a sequence that has the potential to adopt two alternative folding patterns, either a structure involving a pseudoknot, or a simple stem-loop structure. To investigate the structure requirements for efficient frameshifting, mutants in the stem-loop or in the potential pseudoknot regions of a Polish isolate of PLRV (PLRV-P) have been analyzed. Mutations that are located in the second stem (S2) of the potential pseudoknot structure, but are located in unpaired regions of the alternative stem-loop structure, reduce frameshift efficiency. Deletion of the 3' end sequence of the alternative stem-loop structure does not reduce frameshift efficiency. Our results confirm that -1 frameshift in the overlap region depends on the slippery site and on the downstream positioned sequence, and propose that in PLRV-P a pseudoknot is required for efficient frameshifting. These results are in agreement with those recently published for the closely related beet western yellows luteovirus (BWYV).     

An atypical RNA pseudoknot stimulator and an upstream attenuation signal for -1 ribosomal frameshifting of SARS coronavirus

The −1 ribosomal frameshifting requires the existence of an in cis RNA slippery sequence and is promoted by a downstream stimulator RNA. An atypical RNA pseudoknot with an extra stem formed by complementary sequences within loop 2 of an H-type pseudoknot is characterized in the severe acute respiratory syndrome coronavirus (SARS CoV) genome. This pseudoknot can serve as an efficient stimulator for −1 frameshifting in vitro. Mutational analysis of the extra stem suggests frameshift efficiency can be modulated via manipulation of the secondary structure within the loop 2 of an infectious bronchitis virus-type pseudoknot. More importantly, an upstream RNA sequence separated by a linker 5′ to the slippery site is also identified to be capable of modulating the −1 frameshift efficiency. RNA sequence containing this attenuation element can downregulate −1 frameshifting promoted by an atypical pseudoknot of SARS CoV and two other pseudoknot stimulators. Furthermore, frameshift efficiency can be reduced to half in the presence of the attenuation signal in vivo. Therefore, this in cis RNA attenuator represents a novel negative determinant of general importance for the regulation of −1 frameshift efficiency, and is thus a potential antiviral target.     

Crystal structure of a luteoviral RNA pseudoknot and model for a minimal ribosomal frameshifting motif

To understand the role of structural elements of RNA pseudoknots in controlling the extent of -1-type ribosomal frameshifting, we determined the crystal structure of a high-efficiency frameshifting mutant of the pseudoknot from potato leaf roll virus (PLRV). Correlations of the structure with available in vitro frameshifting data for PLRV pseudoknot mutants implicate sequence and length of a stem-loop linker as modulators of frameshifting efficiency. Although the sequences and overall structures of the RNA pseudoknots from PLRV and beet western yellow virus (BWYV) are similar, nucleotide deletions in the linker and adjacent minor groove loop abolish frameshifting only with the latter. Conversely, mutant PLRV pseudoknots with up to four nucleotides deleted in this region exhibit nearly wild-type frameshifting efficiencies. The crystal structure helps rationalize the different tolerances for deletions in the PLRV and BWYV RNAs, and we have used it to build a three-dimensional model of the PRLV pseudoknot with a four-nucleotide deletion. The resulting structure defines a minimal RNA pseudoknot motif composed of 22 nucleotides capable of stimulating -1-type ribosomal frameshifts.     

A heptanucleotide sequence mediates ribosomal frameshifting in mammalian cells.

Ribosomal frameshifting is an essential requirement for replication of many viruses and retrovirus-like elements. It is regarded as a potential target for antiretroviral therapy. It has been shown that the frameshifting event takes place in the -1 direction within a sequence, the slippery sequence, which is usually followed by structured RNA. To distinguish between the basic sequence requirements and the modulating elements in intact cells, we have established a sensitive assay system for quantitative determination of ribosomal frameshifting in mammalian cell culture. In this assay system, the gag and pol genes of human immunodeficiency virus type 1 are replaced by the genes for the functional enzymes beta-galactosidase and luciferase, respectively. The sensitivity of the test system allows us to demonstrate for the first time that the slippery sequence, a heptanucleotide, is sufficient to mediate a basal level of ribosomal frameshifting independent of its position within a gene. The stem-loop sequence serves only as a positive modulator. These data indicate that frameshifting could also occur during translation of cellular genes in which a slippery sequence is present within the reading frame. The resulting putative transframe proteins might have a functional importance for cellular processes.     

Kinetics of ribosomal pausing during programmed -1 translational frameshifting

In the Saccharomyces cerevisiae double-stranded RNA virus, programmed -1 ribosomal frameshifting is responsible for translation of the second open reading frame of the essential viral RNA. A typical slippery site and downstream pseudoknot are necessary for this frameshifting event, and previous work has demonstrated that ribosomes pause over the slippery site. The translational intermediate associated with a ribosome paused at this position is detected, and, using in vitro translation and quantitative heelprinting, the rates of synthesis, the ribosomal pause time, the proportion of ribosomes paused at the slippery site, and the fraction of paused ribosomes that frameshift are estimated. About 10% of ribosomes pause at the slippery site in vitro, and some 60% of these continue in the -1 frame. Ribosomes that continue in the -1 frame pause about 10 times longer than it takes to complete a peptide bond in vitro. Altering the rate of translational initiation alters the rate of frameshifting in vivo. Our in vitro and in vivo experiments can best be interpreted to mean that there are three methods by which ribosomes pass the frameshift site, only one of which results in frameshifting.     

Possible utilization of -1 Ribosomal frame shifting in the expression of a human SEMA6C isoform

We have used bioinformatics approaches to identify a potential case of -1 ribosomal frame shifting in the mRNAs of the three variants of human SEMA6C protein. The mRNAs contain a heptanucleotide slippery sequence followed by a compact H-type pseudoknot. Unlike -1 frameshifting signals in viral or viral-like mRNAs, the slippery sequence and downstream pseudoknot in SEMA6C mRNAs locate 423 nucleotides (encoding 141 amino acids) upstream of the stop codon. The potential -1 frameshifting event would produce a polypeptide of 238 residues encoded by the -1 reading frames. Sequence similarity searches using BLAST indicate that ~90% of the 238 residues match actual protein sequences annotated as SEMA6C proteins in the database. We propose that the mRNAs of human SEMA6C utilize a pseudoknot dependent -1 ribosomal frameshifting mechanism to express novel SEMA6C isoforms.     

An 'elaborated' pseudoknot is required for high frequency frameshifting during translation of HCV 229E polymerase mRNA.

The RNA polymerase gene (gene 1) of the human coronavirus 229E is approximately 20 kb in length and is located at the 5' end of the positive-strand genomic RNA. The coding sequence of gene 1 is divided into two large open reading frames, ORF1a and ORF1b, that overlap by 43 nucleotides. In the region of the ORF1a/ORF1b overlap, the genomic RNA displays two elements that are known to mediate (-1) ribosomal frameshifting. These are the slippery sequence, UUUAAAC, and a 3' pseudoknot structure. By introducing site-specific mutations into synthetic mRNAs, we have analysed the predicted structure of the HCV 229E pseudoknot and shown that besides the well-known stem structures, S1 and S2, a third stem structure, S3, is required for a high frequency of frameshifting. The requirement for an S3 stem is independent of the length of loop 2.     

A Characteristic Bent Conformation of RNA Pseudoknots Promotes –1 Frameshifting during Translation of Retroviral RNA

The structures of four different RNA pseudoknots that provide one of the signals required for ribosomal frameshifting in mouse mammary tumor virus have been determined by NMR. The RNA pseudoknots have similar sequences and assume similar secondary structures, but show significantly different frameshifting efficiencies. The three-dimensional structures of one frameshifting and one non-frameshifting RNA pseudoknot had been determined previously by our group. Here we determine the structures of two new RNA pseudoknots, and relate the structures of all four pseudoknots to their frameshifting abilities. The two efficient frameshifting pseudoknots adopt characteristic bent conformations with stem 1 bending towards the major groove of stem 2. In contrast, the two poor frameshifting pseudoknots have structures very different from each other and from the efficient frameshifters. One has linear, coaxially stacked stems, the other has stems twisted and bent, but in the opposite direction to the efficient frameshifters. Changes in loop size that favor bending (shorter loops) increase frameshifting efficiency; longer loops that allow linear arrangement of the stems decrease frameshifting. Frameshifting pseudoknots in feline immunodeficiency virus and simian retrovirus have different loop sequences, but the sequences at their stem junctions imply the same bent conformation as in the mouse mammary tumor viral RNA. The requirement for a precise pseudoknot conformation for efficient frameshifting strongly implies that a specific interaction occurs between the viral RNA pseudoknot and the host protein-synthesizing machinery.