Viral RNA Pseudoknots: Versatile Motifs in Gene Expression and Replication

REVIEWS

Viral RNA pseudoknots: versatile motifs in gene expression and replication

Ian Brierley*, Simon Pennell‡ and Robert J. C. Gilbert§ Abstract | RNA pseudoknots are structural elements found in almost all classes of RNA. First recognized in the genomes of plant viruses, they are now established as a widespread motif with diverse functions in various biological processes. This Review focuses on viral pseudoknots and their role in virus gene expression and genome replication. Although emphasis is placed on those well defined pseudoknots that are involved in unusual mechanisms of viral translational initiation and elongation, the broader roles of pseudoknots are also discussed, including comparisons with relevant cellular counterparts. The relationship between RNA pseudoknot structure and function is also addressed.

Satellite virus The pioneering work that established pseudoknots genomic RNA serves as the mRNA template for transla- A subviral agent composed of as a genuine folding motif in RNA was carried out in tion and then as a template for replication), pseudoknots an RNA or DNA molecule that the laboratories of Cornelis Pleij, Krijn Rietveld and act in the regulation of initiation of protein synthesis is replicated in association with Leendert Bosch in the early 1980s. These authors were and in template recognition by the viral replicase. By a helper virus, but investigating how it was possible for the 3′ ends of some contrast, in coding regions they modulate the elongation encapsidated in its own encoded capsid protein. plant virus genomes to possess a number of the func- and termination steps of translation. Fewer pseudoknots tional characteristics of transfer RNAs (tRNAs) yet lack have been documented in the mRNAs of viruses with an obvious clover-leaf secondary structure. By applying a DNA genomes, but several DNA bacteriophage mRNAs ‘pseudoknot building principle’, it became clear how these are known to encode pseudoknots16. In the 5′ NCRs, viral RNAs could fold into L-shaped structures resembling these motifs have roles in the regulation of translation tRNAs1,2. A seminal paper from the same authors3 sub- initiation, whereas in the coding region they affect trans- sequently defined the general principles of pseudoknot lation elongation. Pseudoknots have also been described folding and provided the first examples of pseudoknots in in the catalytic RNAs of some RNA satellite viruses, where other RNAs — the central pseudoknot of Escherichia coli they have a role in genome replication17. *Division of Virology, 16S ribosomal RNA (rRNA) and a pseudoknot present Here, we review selected, well characterized examples Department of Pathology, University of Cambridge, in group I introns. of pseudoknots in virus genomes — with an emphasis on Tennis Court Road, Since then, many more pseudoknots have been structure–function relationships — highlighting recent Cambridge CB2 1QP, UK. discovered, and they are associated with a remarkably advances in our understanding of pseudoknot confor- ‡Division of Molecular diverse range of biological activities (Supplementary mation at high resolution and exploiting, where relevant, Structure, National Institute REFS 4–11 for Medical Research, information S1, S2 (tables); reviewed in ). They our improved knowledge of ribosome architecture. The Ridgeway, Mill Hill, are especially associated with key roles in the replication London NW7 1AA, UK. cycles of numerous animal and plant viruses, including, What is an RNA pseudoknot? §Division of Structural in humans, the flavivirus hepatitis C virus (HCV)12, the As defined originally3, a pseudoknot is a structure Biology, Henry Wellcome coronavirus responsible for severe acute respiratory syn- formed upon base-pairing of a single-stranded region Building for Genomic 13 Medicine, University of drome (SARS-CoV) , the oncogenic retrovirus T-cell of RNA in the loop of a hairpin to a stretch of comple- 14 Oxford, Roosevelt Drive, lymphotrophic virus types I and II , and certain strains mentary nucleotides elsewhere in the RNA chain (FIG. 2). Oxford OX3 7BN, UK. of HIV15. Such pseudoknots, referred to as hairpin type (H-type) e-mails: The function of a viral pseudoknot is linked logically pseudoknots, have two base-paired stem regions (S1 and [email protected]; FIG. 1 [email protected]; to its location in the genome ( ; Supplementary S2) and, depending on the number of loop bases that par- 18 [email protected] information S1 (table)). So, in non-coding regions ticipate in the pseudoknotting interaction , two or three doi:10.1038/nrmicro1704 (NCRs) of positive-strand RNA viruses (in which the single-stranded loops (L1, L2 and L3). In most (>85%;

Peptidyl (P) site REFS 19,20) H-type pseudoknots, L2 is absent or very Pseudoknots and translation The site on the small ribosomal short, and the base-paired stems stack coaxially to form Internal ribosome entry. Most eukaryotic cellular subunit that holds the tRNA a quasi-continuous helix. In these structures, L1 spans mRNAs are translated in a cap-dependent manner, with molecule that is linked to the S2 and crosses the deep groove of the helix, whereas L3 the 40S subunit and associated initiation factors scan- growing end of the polypeptide spans S1 and crosses the shallow groove (FIG. 2). ning along the mRNA until the start codon (AUG) is chain. The geometry of the pseudoknot is such that when S2 reached28. Efficient translation also requires mRNA cir- is six or seven base pairs in length, L1 can be as short as a cularization, which is brought about by the interaction of single nucleotide3,21. However, in some pseudoknots, the the 3′-end poly(A) tail-binding protein (PABP) with ini- loops are much longer and include their own secondary tiation factor 4E (eIF4E), bound to the 5′ cap29. However, structure elements. Pseudoknots are also formed upon the genomes of many positive-strand RNA viruses often base-pairing of single-stranded bulge (B), interior (I) lack a cap, a poly(A) tail or both, and translation initia- and multibranched (M) loops with complementary tion involves non-standard mechanisms30,31. regions elsewhere in the RNA (which themselves can One such example is cap-independent internal be constrained in a secondary structure, for example in ribosome entry, in which the ribosome is recruited intramolecular hairpin–loop–hairpin–loop (H–H or internally to a structured region of the mRNA (the so-called kissing loop) interactions)22. However, unless internal ribosome entry site (IRES), usually located all of the loop nucleotides are paired, these pseudoknots in the 5′ NCR) and often directly to the start codon. are generally considered as H-type pseudoknots, as the Pseudoknots have been identified in a number additional base-pairing interaction is viewed as a sub- of IRESs, and their function is best exemplified in structure within the loop. For this reason, the B, I and M the IRESs of the flavivirus HCV and the dicistrovirus nomenclature (as well as H–H) is not extensively used, cricket paralysis virus (CrPV) (FIG. 3). Unlike the mam- and most structures are referred to as H-type pseudo- malian picornaviral IRESs, which retain a requirement knots and often simply as pseudoknots. An additional for many or most canonical initiation factors, the issue regarding nomenclature is loop numbering. In mammalian 40S subunit can associate with the HCV the early pseudoknot literature, most examples did not IRES in the absence of translation initiation factors, possess unpaired residues between the two stems, so the although the formation of the 48S complex (with the convention was to name the groove-spanning loops L1 initiation codon locked into the mRNA-binding cleft and L2 (now L1 and L3). Here, we have opted for the of the small subunit) requires the participation of the L1, L2 and L3 nomenclature, which is more generally eIF2–GTP–Met-tRNAi ternary complex and eIF3 applicable. (reviewed in REF. 32). The HCV IRES forms a defined As will be discussed in more detail below, the biologi- secondary structure that contains two major hairpins cal properties of pseudoknots are intimately linked to (domains 2 and 3) and an essential pseudoknot struc- their structural features4–7,18,23. For example, the geometry ture12 at the base of domain 3 (domain 3e/f) (FIG. 3). of the junction between the stems and the interactions IRES function requires domains 2 and 3, but initial that can occur between the constituent loops and stems binding of the 40S subunit is mediated principally by are often of great functional relevance21,24–27. Indeed, for the basal region of domain 3, including stem–loop many viral pseudoknots, much of the primary sequence 3d, with a modest contribution from the pseudo- is unimportant for function, as long as the conforma- knot33,34. Comparisons of HCV IRES–40S (REF. 35) and tion and overall stability of the structure is maintained. IRES–80S (REF. 36) complexes have revealed that the Where precise nucleotide-sequence requirements have overall appearance of the IRES is similar in the two been identified, this is likely to reflect a specific structural complexes36. In the 80S complex, the pseudoknot cor- necessity, although additional roles might be possible responds to an L-shaped density located at the mRNA (for example, base-specific recognition by proteins). exit channel in the vicinity of ribosomal protein S5 (rpS5) (FIG. 3; Supplementary information S3 (figure)). The assignment of the pseudoknot to the L-shaped Genome replication density is based on molecular modelling and is consistent Internal ribosome entry Genome replication 37 Translation autoregulation Translation regulation with recent crosslinking data . The three-dimensional (3D) structure of the HCV pseudoknot is not available, but in essence it is an H-type pseudoknot in which L1 is 5′ NCRCodingIGR Coding NCR 3′ long and highly structured, and includes the entirety of domains 3a–3e. Why a pseudoknot is present at this key location of the IRES is unclear, but it might be linked to Ribosomal frameshifting Termination codon readthrough a capacity to bind rpS5 (REF. 37). This protein is present at the mRNA exit channel and also associates with IRES Figure 1 | RNA pseudoknots in virus gene expression. A schematic of a generic RNA domain 2 (REFS 36,38). Boehringer and colleagues pro- virus genome is shown. Viral pseudoknots have been described in the 5′ non-coding pose that the HCV IRES domains function synergisti- region (NCR), the coding region, the intergenic region (IGR) and the 3′ NCR, where they function in various steps of the replication cycle. Although the majority of examples are cally to position the AUG into the ribosomal peptidyl (P) 36 from positive-strand RNA viruses, pseudoknots also have a role in the replication cycles site, coupled to movement of the pseudoknot . In this of certain DNA viruses, satellite RNA viruses and viroids. For simplicity, viral pseudoknots model, a conformational change of the four-way junc- involved in long-range interactions (including virus genome circularization) or tion (which includes domains 3a and 3c; FIG. 3) pivoted possessing catalytic activity are not shown, but are discussed in the text. around domain 3d is transmitted to the pseudoknot,

a H–H-type (kissing loop) The three defined domains of this IRES have dis- tinct functional tasks (FIG. 3). Domain 1 contributes to interactions with the 60S subunit in the E- and P-site Multibranched regions, and domain 2 interacts with the 40S subunit at loop ′ Interior loop 3 the E site. Domain 3, which is located predominantly between the A and P sites and is in a similar orientation S2 ′ Bulge loop L1 to ribosome-bound tRNAs, places its most 3 nucleotide triplet (GCU) into the decoding region of the A site11. I-type Here, it binds the anticodon of tRNAAla, which is brought B-type Hairpin loop L2 to the ribosome as part of the ternary complex (elonga- Ala L1 tion factor 1A (eEF1A)–GTP–tRNA ). Subsequently, S2 L3 S1 tRNAAla is pseudotranslocated (without peptide-bond L2 H-type S1 formation) by eEF2 into the P site, allowing delivery of L3 5′ the next tRNA into the A site and authentic elongation 5′ 3′ 41,43,44 M-type H-type pseudoknot to begin . Modelling and structural analysis of the CrPV IRES and IRESs from related viruses, including Plautia stali b 3′ CG intestine virus (PSIV)26,27,45,46, has revealed that the struc- GC (FIG. 3) AU A L1 ture is dominated by three H-type pseudoknots , S2 Deep GC one per domain. Pseudoknot PKI, which essentially GC forms all of domain 3, is characterized by the posses- UA A sion of an AU-rich S1 and short loops. The folding of CG U CG the rest of the IRES is dominated by interactions between A GC (FIG. 3) L3 S1 Shallow pseudoknots PKII and PKIII . The pseudoknot A GC of domain 2, PKIII, is a nested pseudoknot in that it CG C GC is entirely contained within L3 of the pseudoknot of 5′ A C domain 1, PKII. Cryo-electron microscopy (cryo-EM) A A and X-ray crystallography studies26,27 (reviewed in Figure 2 | RNA pseudoknot structure. a | Various structural motifs have been described in REF. 11) have indicated a complex folding strategy that RNA149. Orthodox secondary structures consist of base-paired regions (stems) connected by forces two small hairpins of PKIII, present as substruc- single-stranded loops at stem termini (hairpin loop), or in the body of a stem (bulge (B) or interior (I) loop) or at the junction of several stems (multibranched (M) loop). Pseudoknots are tures in L1 (stem–loop (SL) IV) and L3 (SL V), to project considered as a tertiary structure and form when bases in a loop pair with a single-stranded from a central core and emerge on the same side of the region elsewhere. The hairpin type (H-type) pseudoknot is by far the most common, and this structure to make vital interactions with rpS5 at the tertiary interaction involves bases in the loop of a hairpin loop. The resultant structure E site46–48. Pivotal to this folding strategy are pseudoknot contains two stem regions, S1 and S2, connected by single-stranded loops. In many cases, no loop–helix interactions. In PKIII, for example, an L1–S2 unpaired bases are present between the two stems (L2 is zero), and the stems stack coaxially major-groove interaction positions SL IV, and a second to give a quasi-continuous helix. b | The secondary structure of the pseudoknot of the interdomain interaction occurs between the minor groove ribosomal frameshifting signal of simian retrovirus 1 (SRV-1) is shown alongside three of S2 and four L1 bases of the adjacent pseudoknot PKII, 74 dimensional views of the nuclear magnetic resonance model . The stems are shown as which stabilizes the core. The geometry of the overall fold surface representations and the loops as ribbons (all structural images were prepared using is such that S2 of PKII stacks on S2 of PKIII to create a PyMol). The polarity and handedness of the double helix leads to inequivalence of the loops, with L1 (yellow) crossing the deep groove and L3 (green) crossing the shallow groove. S1 is wedge-shaped section that occupies the mRNA channel blue, S2 is red, L1 is yellow and L3 is green. L2 is not present in the example shown. and directs PKI into the decoding site. Recruitment of the first aminoacyl tRNA to the ribosome requires the participation of eEF2 (REF. 49). The activation of eEF2 has been linked to an IRES-mediated which moves towards the mRNA exit channel, positioning destabilization of a conserved cellular pseudoknot that the AUG correctly into the ribosomal P site and allowing is present in helix 18 of the small subunit rRNA (the subunit joining. The movement could be potentiated by so-called 530-loop pseudoknot; Supplementary infor- eIF3 binding, as this multisubunit initiation factor makes mation S2 (table)). This region of rRNA is involved in intimate contacts with the HCV IRES39. the enhancement of translational accuracy and tRNA 50 Aminoacyl (A) site Pseudoknots also have an essential role in the func- binding , and its destabilization (probably by the pseu- The site on the small ribosomal tion of the intergenic region (IGR) IRES of CrPV40 and doknot of domain 3) is likely to be crucial to the IRES subunit that accepts and other dicistroviruses41. This remarkable IRES, only ~200 mechanism. proofreads incoming aminoacyl nucleotides in length, has been described as an RNA- The structure of the IGR IRESs illustrates how pseu- tRNAs prior to peptidyl 42 transfer. based translation factor because it recruits ribosomes doknots can be used to direct the global folding of an and activates translation without the involvement of RNA sequence. The pseudoknot motif naturally provides Exit (E) site initiation factors or initiator tRNA. The ribosomal 40S the potential for coaxial stacking of constituent helices, The site on the small ribosomal and 60S subunits bind directly to the IRES43, which then but also the opportunity for additional stacking with heli- subunit through which tRNAs pass after they have donated occupies the ribosomal intersubunit space of the 80S ces present in constituent loops. This allows longer helical their amino acid to the growing complex and interacts with key components that form domains to be generated, a common feature in the organ- polypeptide chain. the ribosomal aminoacyl (A), P and exit (E) sites. ization of global RNA structures51. If the pseudoknot is

ab 3b

3a Four-way junction 3c

60S 40S 3e 5′ AUG 3′ 3f Pseudoknot

cd Nested PKIII SL IV 3′ Domain 2 A PKII L3 PKIII L1 A PKIII C 5′ S1 SL V PKIII S2 PKI S2 PKI L1 PKIII L3 PKI L3 PKII L2 PKI L2 PKI S1 PKII S1 Domain 3 Domain 1 40S 60S PKII L1

SL V 25S rRNA L1

SL IV

Domain 1 Domain 2 Domain 2

S5 L11

3′ (to domain 3) Domain 3

Domain 1 18S rRNA Figure 3 | Pseudoknots and internal ribosome entry. a | A secondary structure representation of the hepatitis C virus (HCV) internal ribosome entry site (IRES) with the pseudoknot shown in blue. b | A surface representation of the human 80S ribosome (grey) in complex with the HCV IRES (red) derived from the cryo-electron microscopy (cryo-EM) structure36. Density corresponding to the pseudoknot is indicated in blue. c | A secondary structure representation of the Plautia stali intestine virus (PSIV) IRES is shown above a ribbon representation of the RNA (domains 1 and 2) derived from the crystal structure27. Domain 3 remains to be solved. The secondary structure of the cricket paralysis virus (CrPV) IRES (not shown) is similar. d | A surface representation of the yeast 80S ribosome (grey) is shown in complex with the CrPV IRES (red) derived from the cryo-EM structure26. Below is a fit of the density to the modelled CrPV IRES showing the interactions that occur between the various domains and ribosomal components. Ribosomal proteins are cyan, 25S ribosomal RNA (rRNA) is purple and 18S rRNA is brown.

itself nested in another pseudoknot, additional helical of amino acids (FIG. 4). In retroviruses, frameshifting stacking possibilities are created. Superimposed on this at the overlap of the gag and pol open-reading frames is the capacity of the single-stranded loops to interact (ORFs) allows expression of the viral Gag–Pol poly- with constituent stems to add stability, or with other protein and sets a defined cytoplasmic Gag:Gag–Pol regions of RNA to promote packing of adjacent helices. ratio that is optimized for virion assembly and pack- The compact and complex fold of the IGR IRESs brought aging of reverse transcriptase58. In other RNA viruses, about by the nested pseudoknots is a strategy similar frameshifting allows expression of RNA-dependent RNA to that used to fold certain ribozyme cores, discussed in polymerases (RdRps)59. Maintaining a precise efficiency more detail below. There is no known ribozyme activity of frameshifting has been shown to be crucial to the associated with IRESs, however, which indicates that this replication of HIV-1 (REF. 60) and the retrovirus-like is a general folding strategy that can be used to satisfy double-stranded RNA virus of yeast, L-A61. Similarly, in different mechanistic requirements. other RNA viruses, changing the stoichiometry of non- frameshifted and frameshifted products is also likely to Autoregulation. One of the first viral pseudoknots to be be detrimental. In SARS-CoV, for example, components described16 is encoded by T-even bacteriophages (such as of the viral replication machinery present in the viral T2, T4 and T6) and functions in translational autoregu- polyproteins pp1a and pp1ab (which are expressed by lation of the gene 32 protein (gp32), a single-stranded frameshifting) are predicted to form a heterodimer DNA-binding protein that mainly functions in replica- with a stoichiometry of ~8:1 (REFS 62,63), a ratio that is tion of the viral double-stranded genomic DNA. The consistent with the natural level of frameshifting64,65. For pseudoknot is located some 40 nucleotides upstream of these reasons, frameshifting has emerged as a potential the translation start site (AUG) of the gp32 mRNA and target for antiviral therapeutics. acts as a specific binding site for gp32 itself. At low pro- A typical frameshift signal has two essential elements: tein concentrations, pseudoknot-bound gp32 does not a heptanucleotide ‘slippery’ sequence, at which the ribos- overlap the ribosome-binding site, but as protein levels ome-bound tRNAs slip into the –1 frame, and an adja- increase, cooperative binding of multiple copies occurs, cent mRNA secondary structure, most often an H-type nucleated at the pseudoknot-bound gp32 (REF. 52). This pseudoknot14,66, that stimulates this slippage process assembly blocks access to the Shine–Dalgarno sequence (FIG. 4). How the pseudoknot promotes frameshifting is and so represses translation. Unfortunately, molecular not completely understood, but the mechanism is likely details of the gp32–pseudoknot interaction are lack- to be linked to the helicase activity of the ribosome, ing, but the structure of the pseudoknot isolated from with the pseudoknot presenting an unusual topology bacteriophage T2 has been solved by nuclear magnetic that resists unwinding47,67–71. Takyar and colleagues have resonance (NMR)53. It is a classic H-type pseudoknot shown that the prokaryotic 70S ribosome can itself act with short loops and coaxially stacked stems, albeit with as a helicase to unwind mRNA secondary structures S2 rotated by ~18˚ with respect to S1 to relieve close before decoding70, with the active site located between phosphate–phosphate contacts at the junction while pre- the head and shoulder of the 30S subunit. Prokaryotic serving the stabilizing effects of base stacking. Although ribosomal proteins S3, S4 and S5 that line the mRNA the T2 pseudoknot possesses only a single nucleotide in entry tunnel are implicated in the helicase activity70. L1 (an A), this is stereochemically feasible as the distance Recent cryo-EM images of mammalian 80S ribosomes between the two phosphates across the deep groove of paused at a coronavirus frameshift-promoting pseu- A-form RNA reaches a minimum when six or seven base doknot revealed that the ribosomes become stalled pairs of S2 are bridged3,21. during the trans location phase of the elongation cycle Although the gene 32 system represents the only with eEF2 bound and in the act of transferring peptidyl- Ribozyme known viral example of pseudoknot involvement in the tRNA from the A site to the P site71. Furthermore, the P An enzyme that has an RNA as autoregulation of translation initiation, there are related site tRNA is structurally distorted and in a spring-like it catalytic component. examples in cellular mRNAs (Supplementary information conformation. Consistent with its proposed function, Shine–Dalgarno sequence S2 (table)). For example, translational repression of the the pseudoknot is present at the mRNA entry chan- 54,55 (AGGAGG). This sequence is ribosomal S4 α mRNA operon and autoregulation nel in close proximity to the proteins (rpS3, rpS9 and located 5′ of the AUG codon of ribosomal protein S15 synthesis56 requires specific rpS2) that are likely to form important elements of the on bacterial mRNAs and binding of the respective proteins to pseudoknots in the mammalian 80S helicase (FIG. 4). Namy and colleagues71 functions as the signal for the ′ initiation of protein synthesis. 5 untranslated region (UTR) of their own mRNAs. suggest a mechanical model of frameshifting in which the pseudoknot resists unwinding by the helicase, Transfer messenger RNA Frameshifting. RNA pseudoknots in coding regions compromising translocation by putting tension on (tmRNA). An RNA that are principally associated with sites of programmed –1 the mRNA, leading to bending of the tRNA anticodon possesses both tRNA and ribosomal frameshifting. This is a translational mecha- and, ultimately, repositioning the tRNA on the slippery mRNA characteristics that functions in the recognition nism used by many viruses to coordinately express sequence in the –1 reading frame. The pseudoknot is 7,57 and rescue of ribosomes two proteins from a single mRNA at a defined ratio . also in close proximity to the ubiquitous ribosomal stalled on aberrant mRNAs, During elongation, ribosomes decode the mRNA in regulatory protein RACK1 (receptor for activated C the disposal of the causative triplet steps and the reading frame is accurately main- kinase 1)72. It is not known whether this protein, or defective mRNAs and the promotion of the degradation tained. However, in frameshifting, the ribosome is forced the recruited kinase, has a role in frameshifting, but of ribosome-associated protein to shift one nucleotide backwards into an overlapping this could in principle provide a route to regulation of fragments. reading frame and translate an entirely new sequence frameshifting during virus infection.

a b X-ray crystallography and NMR analysis of ORF1a frameshift-promoting pseudoknots, coupled with func- ORF1b 5′ 3′ tional studies, have revealed features that could account 3′ CG for an intrinsic resistance to unwinding by the ribosomal CGA helicase (FIG. 4). Chief among these is the presence of S2GC L1 AU extensive minor-groove triplex interactions between S1 GC C CG and the crossing L3, first observed in the pseudoknot UG of the luteovirus beet western yellows virus (BWYV; G GC 73 A AU FIG. 4) and subsequently in related luteoviruses and A CG the gag/pro pseudoknot of simian retrovirus 1 (REF. 74). L3 A UGS1 C AU The S1–L3 RNA triplex is likely to be the first feature G UA U GC encountered by the elongating ribosome, and the pres- U GC ence of the ‘third strand’ would conceivably confound ′ GC 5 UUUAAACUGAUAC GC unwinding, at least temporarily. Slippery IBV pseudoknot Another feature of functional importance is the sequence architecture of the junction between the constituent 5′ pseudoknot helices. Several non-canonical interactions have been described in and around the junction, c ′ including base triples, base quadruples and loop–loop 3 Bent tRNA RACK1 Pseudoknot (in the P site) Hoogsteen base pairing as well as distortions such as over-rotation of the stems, helical displacement and rpS3 bending7,75 (FIG. 4). The first NMR study of a frameshift- promoting pseudoknot, from the gag/pro overlap of eEF2 mouse mammary tumour virus, had indeed indicated PK 40S 60S a requirement for a specific bent conformation of the Helix 16 pseudoknot, brought about, in part, by the presence of rpS2 rpS9 an intercalated unpaired adenosine residue between the 76 rpS0 two stems . However, closely related pseudoknots with coaxially stacked stems can clearly stimulate high levels of frameshifting74,77, and it now seems likely that the spe- d cific interactions and resultant architectures of the helical 3′ junction that are required for frameshifting are strongly G12 GC C26 75 S2GC10 A L1 context dependent . Nevertheless, there is no doubt that C26 G12 C the junction conformation is crucial to function and U A GC13 25 A might also present a kinetic or thermodynamic barrier 78,79 GC15 A to unwinding . L3 S1 C5 G C GC A Several cellular homologues of viral frameshift sig- GG A C8 A25 5′ G G 20 nals have been identified and appear to be derived from G 80–82 Figure 4 | Pseudoknots and ribosomal frameshifting. a | The overlapping coding endogenous retroviruses (although some recently 83 sequences open reading frame 1a (ORF1a) and ORF1b of the genome of the identified candidates may have a different origin ). coronavirus infectious bronchitis virus (IBV) are shown above the minimal frameshift- The pseudoknots within these genes are fundamentally promoting sequences of this virus. The pseudoknot promotes frameshifting at the similar in structure to — and retain the functionality of slippery sequence, indicated by a jagged arrow. b | A representation of the stalled, — their viral progenitors, and have a role in the regula- pseudoknot-engaged rabbit 80S ribosome is shown derived from the cryo-electron tion of gene expression during embryogenesis. Another microscopy structure71. The 60S subunit is light grey and the 40S subunit is dark grey. cellular pseudoknot with close structural similarity The peptidyl (P)-site transfer RNA (tRNA) stalled in the complex is coloured turquoise, to viral frameshift pseudoknots is present in bacterial the eukaryotic translocase, elongation factor 2 (eEF2), is purple and the pseudoknot transfer messenger RNA (tmRNA)84–86. This RNA, which structure is red. Below is a schematic of the stalled ribosome. Engagement with the pseudoknot generates a frameshifting intermediate in which the ribosome is stalled contains four pseudoknots (PKI–IV), functions by during translocation with eEF2 bound, generating tension in the mRNA that bends binding to, and ultimately rescuing, ribosomes stalled the P-site tRNA in a (+) sense direction. As a result, the anticodon–codon interaction on damaged mRNAs by a process termed trans trans- breaks over the slippery sequence, allowing a spring-like relaxation of the tRNA in a lation86. PKI of tmRNA shows considerable similarity (–) sense direction. c | A close-up view of the pseudoknot (PK) in the stalled complex, to the BWYV family of pseudoknots, including short with the ribosomal components (rpS0, rpS2, rpS3 and rpS9) in close proximity stems and loops, extensive base stacking, stabilization highlighted. d | The beet western yellows virus (BWYV) pseudoknot73 is illustrated to of the stem junction by a triplex, exclusion of a uridine 70 show examples of features that might confound the ribosomal helicase . Shown from at the junction of the two stems and insertion of L3 into left to right are: a secondary structure model of the pseudoknot, with U drawn to 13 the minor groove of S1 (REF. 84). PKI functions at a dif- indicate its extrusion form the helix; a ribbon representation of the X-ray structure; the L3–S1 (loop 3–stem 1) triplex interaction, with S1 shown as a transparent surface ferent site on the ribosome than frameshift-promoting and the helix within as a purple ribbon; and the BWYV junction quadruple interaction, pseudoknots, however, and its precise role is uncertain. with hydrogen bonds shown as dashed lines. A triplex also forms at the junction of the A growing number of pseudoknots have been shown to two stems (not highlighted). RACK1, receptor for activated C kinase 1. Panels b and c interact with the ribosome, and it is clear that they can are modified with permission from REF. 71 © MacMillan Publishers Ltd. influence function from different sites. This is illustrated

in Supplementary information S3 (figure), in which the accommodates the crossing L1 residues, and L3 is closely location on the ribosome of the pseudoknots involved anchored to S1 through triplex interactions centred in IRES function, frameshifting and trans translation are around the adenosine residue in L3. Indeed, this study compared. was the first to reveal the potential for interplay between Pseudoknots can also promote +1 ribosomal pseudoknot loops and stem regions21. frameshifting. The best characterized examples The tRNA mimicry accounts for the reactivity of the come from the cellular gene ornithine decarboxy- 3′ end of the viral genome with several enzymes that lase antizyme87. However, pseudoknot-dependent +1 recognize tRNA. These include the CCA nucleotidyl frameshifting also appears to be used in the expression of transferase, which adds a 3′ terminal A to complete the certain structural proteins of the phage of Scott A (PSA) 3′ CCA end of the genome upon infection; valyl tRNA bacteriophage that infects Listeria monocytogenes88, synthetase, which aminoacylates the 3′ end with Val; the although this remains the only viral example so far. elongation factor eEF1A, which binds to the TLS to give a viral RNA–eEF1A–GTP ternary complex; and the viral Stop codon readthrough. In the gammaretroviruses, replicase p69/p206 (REFS 96,97). which are typified by murine leukaemia virus (MuLV), An elegant relationship has been unearthed between gag and pol are in the same reading frame and are sepa- the TLS-specific reactivities and the TYMV lifecycle. rated by an UAG stop codon. Here, Gag–Pol synthesis Upon entry into the cell, the 3′ CCA end is completed requires periodic misreading of the stop codon as a sense and aminoacetylated, at which point translation of the (Gln) codon, a process known as termination codon input virus genome, an obligatory step in the produc- suppression or readthrough. Efficient readthrough is tion of the virus replicase, is stimulated synergistically by promoted by an H-type pseudoknot spaced precisely the 5′ cap and the TLS97. The stimulation of translation eight nucleotides downstream of the stop codon89,90. is maximal when the genome is aminoacetylated and is Retroviral readthrough and frameshift signals are thus linked to the formation of a viral RNA–eEF1A–GTP ter- similar in terms of overall organization, with the recod- nary complex97. How eEF1A enhances translation is not ing site (a stop codon or a slippery sequence) separated known, but the available evidence hints at an unexpected from the pseudoknot by a spacer of similar length. There involvement of this elongation factor in the process of is little variation in the size of gammaretroviral pseudo- translation initiation. It has been suggested98 that bind- knots and considerable primary sequence conservation, ing of the viral RNA ternary complex to the A-site of especially in the spacer region and L3 of the pseudoknot. initiating ribosomes could stimulate initiation at the 5′ Many of these bases are functionally essential89,91,92, but end (perhaps in a manner similar to the pseudoknot of have not yet been linked to an explicit structural role92,93. the IGR IRES domain 3 discussed above), but recent No high-resolution structures of these pseudoknots are evidence argues against this specific mechanism99. available as yet, and it remains to be seen whether they Nevertheless, close proximity of the virus genome interact with the ribosome in a manner similar to the ends during translation (the circularization observed pseudoknots involved in frameshifting. However, it is for most cellular mRNAs) could offer an explanation plausible that interactions of the readthrough pseudo- for the translational synergy afforded by the 5′ cap and knot with the helicase or other ribosomal components the 3′ TLS–eEF1A complex. could modulate release-factor access or activity and pro- The TLS of TYMV is also crucial in the switch between mote misreading of the stop codon by the near-cognate translation and replication. As has been elegantly illus- tRNAGln. It has already been shown that sequestration trated for bacteriophage Qβ100 and poliovirus101,102, the of eRF1, in this case as a result of direct binding by movement of ribosomes in the 5′→3′ direction is incom- the MuLV reverse transcriptase, can lead to increased patible with negative-strand RNA synthesis, in which the readthrough94. RNA polymerase travels 3′→5′. Thus, following an initial burst of translation, the viral mRNA must be cleared of Pseudoknots in the 3′ NCR ribosomes to allow replication from the 3′ end. Negative- Translation and replication. Several positive-strand strand synthesis in TYMV is initiated from the second C RNA viruses harbour pseudoknots in the 3′ NCRs of the 3′ CCA triplet following pseudoknot-dependent (Supplementary information S1 (table)). The best studied binding of the replicase to the TLS103,104. It has been dem- examples come from plant viruses and, in particular, the onstrated recently that this reaction is inhibited by the pseudoknot that induces the formation of the tRNA-like binding of eEF1A to the valylated TLS105. So, translation structure (TLS) at the end of the genome of turnip yel- is favoured until the levels of viral RdRp are sufficient to low mosaic virus1,2 (TYMV; FIG. 5). This short H-type compete with eEF1A for binding to the TLS or perhaps pseudoknot forms part of the aminoacyl acceptor arm until genomes are sequestered into vesicular sites of virus of the TLS, stacking against an adjacent hairpin (itself replication, which might be free of competing eEF1A the equivalent of the tRNA T-stem–loop) to generate a and aminoacyl tRNA synthetases. The TLS might also quasi-continuous helix that mimics the acceptor arm of be involved in genome packaging: in the related brome tRNA and that can be adenylated and aminoacylated mosaic virus, viral RNAs lacking the TLS fail to assemble with Val95. NMR studies of this region of the TLS have into virions106. revealed that the single-stranded loops of the pseudo- In TYMV, a second pseudoknot is present imme- knot introduce minimal distortion in the A-form helical diately upstream of the TLS, and this pseudoknot also shape of the molecule. The major groove comfortably contributes to translational enhancement, probably by

ab3′ A C C 3′ A A C A AGGCU U C G C U C UCCGAG CG U G 5′ A UA U G C U U U C G G C C G C A U C C U A U U 5′…CCU UAAAAUCGU C A C C A U A U GCCCU U A C U A A G GACAC A A G CGGG D A U G CCGCUC U UGC D CUCGA CUGUG G A T ΨC U GCGAG A G AGAGC C U A U AU GG G U C G C G AGC U A C G G C A U U A G C CG A Ψ C C C A C A U Y C AC G A A Acceptor arm L3 UCA C S2 A UCCCG CCC UCGGAACCA 3′ Acceptor arm T-like loop A A U C 3′ GGGC GGG AGCCU CACAGAAUUCGCACCA CGU T loop G S1 C GUGUCUUAGGCG ′ U G CUUU ΨT 5 D-like loop U A L1 G D A C G D U G C D loop G G C G A A C A G C G C U L3 A U A U G UAAA S2 G C G C G ′… G C G A 5 CCUAAG AUCGUUAG U AUAAU G UUC UAGC C G C G S1 U A C G UUCU G C A U L1 U A G C Anticodon arm C G A Ψ C C Anticodon arm C A C A U Y C A C G A A

UPK TLS Figure 5 | Pseudoknots and transfer RNA-like structures. a | The 3′ end of the turnip yellow mosaic virus (TYMV) genomic RNA. In the upper panel, the predicted secondary structure is shown, with pseudoknotting interactions indicated by dashed red lines. Below is a secondary structure representation of the folded molecule, showing the transfer RNA (tRNA)-like structure (TLS) and the pseudoknots in the acceptor arm and upstream of the TLS (UPK). The ribbon representation (boxed) is derived from the nuclear magnetic resonance structure of the acceptor arm pseudoknot21. b | For comparison, secondary structure representations of tRNAPhe are also shown. D, dihydrouridine modified bases; T, ribothymidine base.

acting as a spacing element to present the functional Despite the complexity of folding of the TMV TLS, TLS to enzymes97 (FIG. 5). The presence of such upstream the UPD seems to have usurped its role, at least in terms pseudoknots is not uncommon and indeed, tobamo-, of translational enhancement110. Although the TLS can hordei-, furo-, pumo- and certain tymoviruses boast be aminoacetylated with His and can bind eEF1A22, the clusters of pseudoknots (between two and seven) in this major player is the UPD, in which the highly conserved region107, which is termed the upstream pseudoknot PKII and PKI can be crosslinked to eEF1A, independ- domain (UPD). So, in tobacco mosaic virus (TMV), ently of TLS aminoacylation111, and form an essential for example, a total of five pseudoknots are present in element of the promoter for negative-strand synthesis112. the 3′ UTR, three of which form the quasi-continuous The UPD can also bind to the heat-shock chaperone double helical stalk of the UPD (5′ PKIII, PKII, PKI 3′) HSP101, although the protein does not appear to be and two of which (5′ PKb, PKa 3′) are present in the essential for efficient replication of TMV113. It seems that TLS108. In the TLS, PKa forms part of the acceptor arm there is a general requirement for the binding of certain and PKb forms the central core, imposing the tRNA- cellular proteins to the 3′ end that does not necessarily like shape and orienting the UPD109. PKb is a B-type have to be mediated solely by a TLS. pseudoknot and forms a Y-shaped three-way junction Another model system used to study the role of viral that is proposed from modelling to have some struc- NCR pseudoknots in facilitating translation, replication tural similarity to the ribozyme of hepatitis delta virus and the switch between the two is tomato bushy stunt virus (HDV; see below), although it displays no catalytic (TBSV)114–117. The TBSV genome is uncapped and lacks a activity109. poly(A) tail. It recruits the translation machinery initially

Box 1 | Computational prediction of RNA pseudoknots Pseudoknots in non-coding regions have also been documented in animal RNA viruses, with the vast majority RNA secondary structure prediction is not trivial and is restricted by our incomplete known to be essential for virus replication (Supplementary understanding of RNA thermodynamics and folding kinetics, and by long computer information S1 (table)). Mostly, they function in transla- 137 processing times . RNAs can also adopt alternative conformations. Most tion, genome replication or the switch between the two. programmes determine a minimum free-energy structure from the primary sequence, but cannot take into account pseudoknot topology, predominantly owing to the However, they are not well characterized structurally (at computational complexity138. Indeed, it has been calculated that to find a general high resolution) and few details of the molecular interac- method to search sequences for minimum free-energy pseudoknots is an intractable tions in which they participate are available. task139. Nevertheless, a substantial proportion (perhaps a quarter) of the pseudoknot literature deals with computational approaches to pseudoknot identification. Most Pseudoknots and ribozymes commonly, a heuristic approach is taken where the search is restricted to certain Hepatitis delta virus. HDV is a satellite RNA virus of pseudoknot types. However, most programmes are effective only for short sequences, humans that replicates in association with a helper virus, as processing time can increase as the third to sixth power of sequence length, hepatitis B virus17. The circular, single-stranded RNA depending on the algorithm used. Another approach that has been used successfully genome of HDV is replicated through an intermedi- is to perform simulations of RNA folding. These methods do not search for minimum ate, the antigenome, by a double rolling-circle mechanism free-energy structures and are therefore computationally more efficient. For longer sequences, a more efficient approach is to use pattern matching to perform a primary that requires self-cleavage by closely related genomic screen of a sequence database to gather those sequences with the potential to form and antigenomic versions of a ribozyme. The HDV pseudoknots of a defined type and subsequently analyse only these sequences. ribozymes have been extensively studied as a model for Multiple sequence comparisons are also of use. A combination of approaches is the mechanism of catalytic RNAs9 and fold into similar usually needed to have confidence in the outcome of a database search and the structures, characterized by a nested double pseudoknot pseudoknots predicted within. The capacity of the programme to deal with that helps form the catalytic site and brings great stability pseudoknots containing stem regions with non-Watson–Crick pairs or bulged to the RNA118 (Supplementary information S4 (figure)). residues is often an issue. Despite these limitations, pseudoknot search programmes The nested double-pseudoknot motif is also common are a valuable resource. Examples of commonly used programmes and webservers are in cellular ribozymes (Supplementary information S2 provided in TABLE 1 (see also REF. 140 for a review). (table)), for example, the HDV-like ribozyme present in an intron of the gene that encodes human cytoplasmic polyadenylation binding protein 3 (REF. 119) and the to the 3′ NCR, a process requiring a highly structured glmS riboswitch ribozyme present in the 5′ UTR of the 3′ cap-independent translational enhancer (3′ CITE), gene that encodes glucosamine-6-phosphate synthase comprising a Y-shaped hairpin domain upstream of a in numerous Gram-positive bacteria120,121. The intricate 3′-end pseudoknot. It is thought that upon infection, connectivity that results from nesting two pseudoknots the pseudoknot is folded (the closed conformation) and within each other makes it possible for these short RNAs translation factors gather on the 3′ CITE. Translation to adopt complex and stable 3D folds. initiation complexes are then transferred to the vicinity of the 5′ NCR by virtue of genome circularization, A viral telomerase pseudoknot mediated, at least in part, by a kissing-loop interaction Telomerases are ribonucleoprotein complexes responsi- between a stem–loop in the 3′ CITE and a partner ble for the maintenance of telomeres10. In addition to a close to the 5′ end of the genome (SL III). Translation specialized reverse transcriptase (TERT), an RNA com- complexes then scan to the first AUG and begin protein ponent (telomerase RNA (TR)) is present and includes synthesis. Subsequently, the pseudoknot at the 3′ end of the RNA template for telomere addition and a highly the genome is destabilized, possibly by binding of the conserved pseudoknot necessary for telomerase activity. accumulating viral RdRp, yielding an ‘open’ complex that Like frameshift-promoting pseudoknots, the TR pseudo- is compatible with negative-strand synthesis117. Another knot has extensive triplex interactions at the junction of pseudoknot (PK-TD1) in the 5′ NCR is also required for the two stems25 (Supplementary information S5 (figure)). efficient replication, although its mechanism of action is It has been proposed that the pseudoknot region makes not fully understood. contacts with the TERT122 and is needed for telomere

Table 1 | Examples of pseudoknot prediction programmes Task Programme Operating system URL Pattern matching RNAmotif Linux http://www.scripps.edu/mb/case/casegr-sh-3.5.html Pseudoknot prediction PknotsRG Linux, OSX, Windows, Web server http://bibiserv.techfak.uni-bielefeld.de/download/tools/ from short sequences pknotsrg.html Pseudoknot prediction HotKnots Linux http://www.cs.ubc.ca/labs/beta/Software/HotKnots from short sequences Pseudoknot prediction HPKNOTTER Web server http://bioalgorithm.life.nctu.edu.tw/HPKNOTTER from longer sequences Pseudoknot visualization PseudoViewer Web server, Windows http://pseudoviewer.inha.ac.kr Pseudoknot database PseudoBase Web server http://biology.leidenuniv.nl/~batenburg/PKBGetS1.html

Box 2 | RNA pseudoknot aptamers Aptamers are small nucleic acid ligands that are generated in vitro against various biological molecules. The majority are produced using the SELEX (selective evolution of ligands by exponential enrichment) method141. A pool of randomized RNAs is selectively enriched over repetitive rounds of target binding and subsequent sequence amplification until the surviving pool consists of sequences that bind to the target with high affinity and specificity. Initial experiments with SELEX focused on viral polymerases, confirming the sequence selectivity of T7 DNA polymerase141, and these were followed by the isolation of pseudoknotted aptamers able to bind to HIV-1 reverse transcriptase142. These aptamers have been shown to bind with picomolar affinity143 and can block HIV replication in tissue culture144, inhibiting the polymerase at all stages of genome replication145. The crystal structure of aptamer-bound reverse transcriptase shows how the pseudoknot ligand (coloured strand) is bound along the cleft between the polymerase active site and the RNAse H domain146 (see image; stem 1 (S1) is blue, S2 is red, loop 1 (L1) is yellow, L3 is green and additional non-pseudoknot bases are orange; dark grey is p51 and light grey is p66). The pseudoknot holds the polymerase in a ‘closed’ conformation, blocking its action by competitive inhibition of the primer–template binding site. Because of their specificity, there is considerable interest in the use of aptamers as therapeutics. However, aptamers cross cell membranes inefficiently owing to their hydrophilicity and are mostly restricted to extracellular applications such as the blocking of viral glycoproteins to prevent cell attachment and viral entry147. To be effective, such aptamers need to be chemically modified so as to resist host ribonucleases. SELEX experiments regularly isolate pseudoknotted ligands148, presumably reflecting the range of binding surfaces that pseudoknots can offer.

repeat-addition processivity123. Indeed, a switch between non-Watson–Crick interactions73 and are more stable the pseudoknot and a partially unfolded form might be than an equivalent hairpin (containing a base-paired stem important in the translocation of telomerase during tel- that is equivalent to S1 plus S2). Thus, a very stable motif omere addition124–126. High levels of telomerase activity are can be included in an RNA when space — either in terms detected in a large range of cancers and are closely associ- of genomic coding capacity or molecular dimensions ated with the immortalization process127,128. Recently, a — is at a premium. At the other end of the spectrum, chicken TR homologue, vTR, has been described in the many pseudoknots are considerably less stable than their oncogenic herpesvirus Marek’s disease virus129. Deletion equivalent hairpin and could conceivably act as regulatory of the two copies of vTR from the virus genome led to switches, oscillating between stem–loop and pseudoknot Rolling-circle replication 132 A mode of replication that reduced incidence and severity of lymphoma in infected conformations in response to environmental signals . uses a circular molecule as chickens, indicating that vTR has the attributes of an Pseudoknots also offer binding sites for proteins or single- a template to produce oncogene130. An understanding of how vTR supports stranded loops of RNA. The often extensive intra- and concatemers of linear lymphomagenesis in genetic and molecular terms has the intermolecular contacts that pseudoknots engage in molecules. potential to yield new insights into telomerase function provide many targets for such interactions. Indeed, the 131 Riboswitch in cancer . in vitro selection of RNA aptamers that bind various bio- A conformational switch in an molecules often generates pseudoknotted RNAs (BOX 2). RNA molecule that is induced The versatile pseudoknot Pseudoknotting can also be the most efficient way of by a small metabolite, and that The development of sophisticated algorithms (BOX 1; folding RNAs in an active conformation (for example, leads to a switch in gene- regulatory function. TABLE 1) to scan genomic sequences for RNA structural ribozymes). motifs has highlighted the prevalence of the pseudoknot Long-range interactions are also facilitated by this Aptamer fold in the RNA universe, indicating evolutionary selec- motif to organize global folding (for example, in the ribos- An RNA domain, either tion. A plausible explanation for this abundance is that ome itself; Supplementary information S2 (table)) and to engineered or natural, that forms a precise 3D structure pseudoknots offer functional versatility. Some pseudo- link separate domains of RNA together. In bacteriophage and selectively binds a target knots (for example the BWYV frameshift pseudoknot) Qβ, although the viral replicase is anchored at a site some molecule. are extensively stabilized by both Watson–Crick and 1.2 kb from the 3′ end of the genome where replication

initiates, an adjacent pseudoknot forms a long-range about the structure and function of pseudoknots. Until interaction that brings the 3′ end to the active site of the recently, atomic resolution structural information has polymerase133. Such connections also offer the potential been restricted to short, often very stable, pseudoknots. for global regulation of gene expression. In barley yellow Elucidating the cryo-EM and crystal structures of the dwarf virus, L3 of the frameshift-promoting pseudoknot IGR IRESs of CrPV26 and PSIV27 were huge steps for- is over 4 kb in length (of a 5.6-kb genome) and links the ward, but similar monumental efforts will be required to frameshift region with the 3′ end of the genome134. It has solve those structures in which the pseudoknot orches- been suggested that disruption of this long-range contact trates the folding of long and complex domains, as is is a means to regulate ribosome and replicase traffic on found in many viral NCRs. Several viral pseudoknots the viral genome135. remain poorly characterized, both in terms of structure Especially in RNA viruses, there is often enormous and biological activity. A number of these have been selective pressure, and genomes that are optimized for shown to interact with proteins, both viral (including viral fitness rapidly dominate. Although it is plain that RdRp) and cellular, yet molecular details are lacking. some or all of the features of pseudoknots discussed above Furthermore, we do not know whether pseudoknots are could be reproduced in other ways, it might be that this ever substrates for viral or cellular helicases present in can only be achieved at considerable cost to fitness. infected cells; such activities could offer an extra level of control of virus gene expression and replication. The use Future perspectives of pseudoknots in antiviral strategies should also be con- Although 25 years have passed since the first pseudo- sidered more broadly. Antisense oligomers targeting the knot was described1, much remains to be discovered. SARS frameshift-promoting pseudoknot have marked Foremost among the tasks ahead is to determine the true antiviral activity136, and pseudoknotted aptamers have frequency of these motifs in natural RNAs. The majority been shown to block HIV replication (BOX 2). of pseudoknots described in this article were identified The study of viral pseudoknots will continue to excite as part of the normal process of scientific research, but and challenge researchers. It is unquestionable that computational approaches to their identification will many more pseudoknots remain to be discovered, and be of increasing relevance. To maximize the proportion it is highly likely that some of these will possess novel of genuine candidate pseudoknots identified in such activities or have unprecedented functions. The field has screens, a better understanding of the thermodynamic come a long way since the early experiments of Pleij and parameters that govern pseudoknot formation will be his collaborators, yet there is still much to be discovered invaluable. Additionally, we will need more information about these fascinating motifs.

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