key: cord-0821163-1jorjnwi
authors: Bhatt, Pramod R.; Scaiola, Alain; Loughran, Gary; Leibundgut, Marc; Kratzel, Annika; McMillan, Angus; O’ Connor, Kate M.; Bode, Jeffrey W.; Thiel, Volker; Atkins, John F.; Ban, Nenad
title: Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome
date: 2020-10-26
journal: bioRxiv
DOI: 10.1101/2020.10.26.355099
sha: 63f7a71522051dfd6ec42e50a9f32a18a344a4d2
doc_id: 821163
cord_uid: 1jorjnwi

Programmed ribosomal frameshifting is the key event during translation of the SARS-CoV-2 RNA genome allowing synthesis of the viral RNA-dependent RNA polymerase and downstream viral proteins. Here we present the cryo-EM structure of the mammalian ribosome in the process of translating viral RNA paused in a conformation primed for frameshifting. We observe that the viral RNA adopts a pseudoknot structure lodged at the mRNA entry channel of the ribosome to generate tension in the mRNA that leads to frameshifting. The nascent viral polyprotein that is being synthesized by the ribosome paused at the frameshifting site forms distinct interactions with the ribosomal polypeptide exit tunnel. We use biochemical experiments to validate our structural observations and to reveal mechanistic and regulatory features that influence the frameshifting efficiency. Finally, a compound previously shown to reduce frameshifting is able to inhibit SARS-CoV-2 replication in infected cells, establishing coronavirus frameshifting as target for antiviral intervention.

Programmed ribosomal frameshifting is the key event during translation of the SARS-CoV-2 RNA genome allowing synthesis of the viral RNA-dependent RNA polymerase and downstream viral proteins. Here we present the cryo-EM structure of the mammalian ribosome in the process of translating viral RNA paused in a conformation primed for frameshifting. We observe that the 5 viral RNA adopts a pseudoknot structure lodged at the mRNA entry channel of the ribosome to generate tension in the mRNA that leads to frameshifting. The nascent viral polyprotein that is being synthesized by the ribosome paused at the frameshifting site forms distinct interactions with the ribosomal polypeptide exit tunnel. We use biochemical experiments to validate our structural observations and to reveal mechanistic and regulatory features that influence the 10 frameshifting efficiency. Finally, a compound previously shown to reduce frameshifting is able to inhibit SARS-CoV-2 replication in infected cells, establishing coronavirus frameshifting as target for antiviral intervention.

Ribosomal frameshifting, a process during which the reading frame of translation is changed in the middle of the coding sequence, is one of the key events during translation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive sense single-stranded RNA genome.

This so-called programmed -1 translational frameshifting is conserved in all coronaviruses and is 5 necessary for synthesis of viral RNA dependent RNA polymerase (Nsp12 or RdRp) and downstream viral non-structural proteins encoding core enzymatic functions involved in capping of viral RNA, RNA modification and processing, and RNA proof-reading. Although the translational machinery typically prevents frameshifting as a potential source of one of the most disruptive errors in translation (1, 2) , many viruses rely on programmed ribosomal frameshifting 10 to expand and fine-tune the repertoire and stoichiometry of expressed proteins (3) .

Programmed -1 frameshifting in SARS-related coronaviruses occurs at the slippery sequence U_UUA_AAC in the context of a 3' stimulatory RNA sequence that was predicted to form a 3-stemmed pseudoknot structure (4), the importance of which was tested by our lab and others (5, 6) . The frameshifting occurs with high efficiency (25-75% depending on the system 15 used (5) (6) (7) (8) (9) and changes the reading frame to UUU_AAA_C (10) (Fig. 1A) . A putative secondary structure element in the viral RNA that forms a loop upstream of the shift site has been proposed to play an attenuating role in frameshifting and is referred to as the 5' attenuator loop (11) .

Maintaining the precise level of coronavirus frameshifting efficiency is crucial for viral infectivity, evidenced by the remarkable fact that mutation of a single nucleotide in the 20 frameshifting region of the SARS-CoV RNA results in a concomitant abrogation of viral replication (12). Therefore, the criticality of pseudoknot-dependent -1 ribosomal frameshifting in the propagation of SARS-related coronaviruses, a process that does not occur in human cells, presents itself as an opportune drug-target with minimal tolerance for drug-resistant mutations.

Due to its importance in the life cycle of many important viruses and coronaviruses in 25 particular, programmed frameshifting has been extensively studied using a range of structural and functional approaches (3) . The structure of a 3' stimulatory pseudoknot in isolation or in context of the viral genome has been proposed recently by various groups using techniques that include molecular dynamics, nuclease mapping, in vivo SHAPE, NMR and cryo-EM (6, (13) (14) (15) (16) .

Furthermore, a ribosomal complex with a frameshift stimulatory pseudoknot from the avian 30 infectious bronchitis virus was reported at low resolution (17) . Here, to provide a structural and mechanistic description of the events that ensue during ribosomal frameshifting, we investigated mammalian ribosomes captured in distinct functional states during translation of a region of SARS-CoV-2 genomic RNA where -1 programmed frameshifting occurs. 5 

We initially intended to capture a 0 frame, pre-frameshift ribosomal complex by first introducing a stop codon in place of the second codon of the slippery site (U_UUA_AAC to U_UUA_UAA), achieving stalling by means of utilizing a mutant eRF1 (AAQ) that is unable to release the nascent polypeptide and would, therefore, trap the ribosome in the act of decoding the mutant 10 frameshift site in the 0 frame. Translating complexes were prepared in an in vitro translation reaction using a rabbit reticulocyte lysate (RRL) system generated in-house (see Methods). The ribosomes were programmed with mRNA harboring a region of the SARS-CoV-2 genome that encodes proteins Nsp10 (C-terminus), Nsp11 and the majority of Nsp12. Western blotting showed that when using the WT RNA template, frameshifting was efficient, while the stop 15 codon mutation prevented frameshifting and led to ribosome stalling when eRF1 (AAQ) was present (Fig. 1B) . Curiously, we also observed a prominent band corresponding to a tRNAbound product paused in the vicinity of the frameshift site even in the absence of eRF1 (AAQ).

Indeed, cryo-electron microscopic 3D reconstruction of RNCs purified from the reactions supplemented with eRF1 (AAQ) revealed that the majority of ribosomal particles corresponded 20 to a paused ribosome with no detectable eRF1 (AAQ) (fig. S1). The 80S ribosomes were found captured in the process of translation with P-and E-site tRNAs bound, and with a nascent polypeptide chain in the ribosomal tunnel.

Incidentally, the 2.2 Å resolution obtained for our reconstruction allowed us to build the most accurate structure of a mammalian 80S ribosome so far and directly visualize virtually all 25 rRNA modifications proposed for the human ribosome based on quantitative mass spectrometry, which is not surprising since all modified residues are also conserved in rabbit rRNAs (18) Importantly, further classification revealed a prominent density for a complete 3' frameshifting stimulatory pseudoknot at the entry of the mRNA channel on the 40S subunit ( Fig.   1C -D). The resolution of this reconstruction ranged from 2.3 Å at the core of the ribosome to ~7 Å at the periphery, where the most flexible regions of the pseudoknot are located ( fig. S1 and   S2 ). Based on the high-resolution maps that allowed visualization of the codon-anticodon 5 interactions and modifications in the tRNA (Fig. 1E, fig. S4A -B), we could unequivocally determine that a Phe-tRNA(Phe) was bound at the P-site (20) . This implied that the ribosome is paused by the downstream pseudoknot such that the P-site tRNA interacts with the UUU codon just prior to the first codon of the frameshift site ( Fig. 2A ). This corkscrew-like formation provides a bulky and well-structured obstacle wedged at the mRNA entry channel, having the potential to resist unwinding by the helicase activity of the ribosome and generating tension on the upstream mRNA up to the decoding center. Stem 1 of 20 the pseudoknot forms a 9 bp helix which is GC rich at the bottom (Fig. 1F ). The penultimate nucleotides of the 'spacer region' prior to Stem 1 are located at the mRNA entry tunnel, where they interact with several basic residues in the C-terminal domain of uS3 on one side and are supported by uS5 from the other, with an additional weak contact being made from the Cterminal end of eS30. uS3 and eS30 are primary components of the ribosome helicase and uS5 25 has been proposed to be a component of the ribosomal helicase processivity clamp at the mRNA entry site (22, 23) . The observed distance between the P-site UUU codon and Stem 1 of the pseudoknot underscore the critical dependence of the frameshifting efficiency on the length of the spacer region (24) . Translocation to the next codon would place the frameshifting codon UUA into the P-site, with a simultaneous increase in the tension of the mRNA and unwinding of the GC-rich base of Stem 1 upon entering into the mRNA entry channel.

The pseudoknot structure also reveals a hitherto unobserved and possibly unappreciated role for the distal site of the mRNA entrance channel in helicase activity. While mRNA unwinding studies outside the mRNA entrance channel have so far implicated only a helix in the 5 C-terminal domain of uS3 (25), we notice that Loop 1 of the pseudoknot contacts the N-terminal domain of uS3 as well as the C-terminal tail of eS10 ( Fig. 2B and fig. S4D ), whereas the flippedout base G13486 in this loop forms specific interactions (Fig. 2B) . Furthermore, as the pseudoknot is located at the entry to the mRNA channel, helix h16 of the 18S rRNA is noticeably pushed outwards due to a direct contact with the minor groove of Stem 1 (Fig. 2B, fig.   10 S5A). Since the pseudoknot wedges between the head and the body of the small ribosomal subunit, it would restrict their relative motions that need to take place during translocation. This is consistent with the studies on dynamics of coronavirus frameshifting, which revealed that the mechanism of -1 frameshifting involves restriction of small subunit head motion (26) .

The structure also reveals another key aspect of the architecture of the pseudoknot as the 15 ribosome encounters it. The start of the pseudoknot is shifted relative to the predicted secondary structure (13) (14) (15) (16) 21) by two nucleotides. The two opposed nucleotides, which were assumed to base pair with Stem 1, are actually forming the start of Stem 3 by pairing with bases predicted to be in the single-stranded linker 2 (Fig. 1F, fig. S5B -C). Our cryo-EM density reveals that Loop 3 accommodates a total of 4 nucleotides, three of which were originally attributed to Stem 2. Thus, 20 we observe that Loop 3 is shifted and expanded relative to the initially predicted secondary structures (13) (14) (15) (16) 21) .

To functionally support our structural findings and confirm the nature and specificity of the pseudoknot interactions, we performed structure-guided mutagenesis experiments using dual luciferase reporter assays in HEK cells (see Methods) and monitored the frameshifting efficiency 25 relative to the WT (Fig. 2C) . Mutation of G13486 of Loop 1 to another purine reduced the frameshifting efficiency to 30% of the WT level, and mutation of this base to a pyrimidine further reduced frameshifting to 15%. As expected from our structural data, deletions of the nucleotides of the spacer regions also had a deteriorating effect on frameshifting. Loss of Loop 1 entirely abolished frameshifting. Deletion of a single nucleotide of Loop 3, which would not be expected to have an effect on the folding of the pseudoknot based on the previously suggested secondary structures where it is flipped out (21) , diminished the frameshifting rate to 25% of the WT level. Loss of the entire Loop 3 reduced frameshifting to 10% of WT levels.

Frameshifting efficiency depends on the position of the "0" frame stop codon 5 In SARS-CoV-2, the 0 frame stop codon is located 5 codons downstream of the frameshift site and is a constituent of Stem 1. The placement of the stop codon in such proximity to the frameshift site is a common feature in coronaviruses, and its presence in a critical region of the stimulatory pseudoknot prompted us to probe the effect of the distance of the 0 frame stop codon on frameshifting. To this end, knowledge of the 3D structure of the pseudoknot helped us to 10 confidently manipulate the stop codon without hampering pseudoknot formation. We introduced mutations to incrementally extend the stop codon from the WT position and to completely remove the occurrence of a stop codon in the 0 frame (Fig. 2D ). While introducing a stop codon 6 nucleotides downstream of the WT position only marginally decreased the frameshifting rate (98% of WT), a stronger attenuation was observed when the distance of the stop codon was 15 increased to 15 nucleotides from the WT stop (80% of WT). Finally, removal of the stop codon by two different point mutations led to a reduction of frameshifting efficiency to 45% of WT levels.

Taken together, these observations suggest that the stop codon position plays an important role in maintaining optimum frameshift efficiency. We propose that the stop codon 20 serves to prevent the closely trailing ribosome from encountering an RNA that was unfolded by the leading ribosome. In this case, upon encountering a stop codon, termination and subunit disassembly will occur, which will provide an opportunity for the pseudoknot to refold without the constrains of the mRNA channel (see Conclusions). This mechanism, consistent with our biochemical data, increases the efficiency of frameshifting to the levels required by SARS-CoV- 25 2 and may be used by viruses in general when high-efficiency frameshifting is required.

The extreme sensitivity of the coronavirus to the finely controlled frameshifting levels (12) may present an opportunity to develop compounds that interfere with the frameshifting process and thus inhibit replication of the virus. Using computational modeling and reporter assays, 5 compounds that have been predicted to bind the pseudoknot and inhibit SARS-CoV-2 frameshifting were described (21, 27) , but never tested with respect to their ability to inhibit viral replication. To demonstrate that the inhibition of frameshifting is a plausible strategy for drug development, we synthesized MTDB, one of these previously described compounds (21, 27, 28) , and tested whether it is able to reduce viral levels in infected African green monkey Vero 10 cells (Fig. 3 , see Methods). The compound showed no cellular toxicity and resulted in a 3 orders of magnitude reduction of SARS-CoV-2 titer, with the half maximal inhibitory concentration (IC50) of 48 µΜ (Fig. 3) . Although this potency range is still far from what would be expected from a potential drug candidate, it nevertheless provides a starting point for high-throughput screening, and establishes that frameshifting is a viable target for therapeutic intervention against 15 SARS-CoV-2.

Strikingly, in the reconstruction of the paused translating ribosome, the nascent chain that corresponds to the viral polyprotein was visible along the entire length of the ribosomal exit 20 tunnel (Fig. 4A) . The density corresponded to the C-terminal region of Nsp10, which is the activator of the viral proofreading exonuclease and N7-methyltransferase Nsp14 (29, 30) and then, depending on the frameshifting event, continues as either the viral RNA-dependent RNA polymerase Nsp12 (5) or protein Nsp11, the function of which is yet unknown (Fig. 1A and 4B ).

The nascent chain makes several specific interactions with the ribosomal tunnel, one of which is 25 at the constriction site where arginine 4387 of Nsp10 interacts with A1555 of the 28S rRNA (corresponding to A1600 in humans, numbering according to PDB 6EK0 (19)) and is stabilized by the preceding leucine 4386 (Fig. 4C) . Interestingly, these two amino acids are very well conserved across multiple coronaviruses ( fig. 4G ), although they are located in the unstructured C-terminal region of Nsp10 and therefore considered not to be important for the fold of the protein (31) .

Further down the tunnel, the C-terminal end of Nsp10 adopts a partially-folded zinc finger motif (Fig. 4D-E) , which upon superposition reveals similarity with the corresponding fully folded C-terminal domain previously observed in the crystal structure of SARS-CoV Nsp10 5 (31) . Tryptophan 4376 located between the two pairs of cysteines that form the zinc finger stacks with A2261 (A2418), an interaction that might serve to promote the change of nascent chain direction and facilitate folding of the zinc finger at the end of the exit tunnel. Co-translational events, such as insertion of a transmembrane domain at the exit of the ribosomal tunnel, was shown to promote -1 ribosomal frameshifting in alphaviruses (32). 10 To investigate whether the observed contacts between the nascent chain and the ribosomal tunnel are specific, and whether these interactions and co-translational folding of Nsp10 might play a role in modulating the frameshifting process, we employed our dual luciferase reporter assay to measure the frameshifting efficiency of WT and mutant nascent chain sequence constructs. As our measurements in HEK cells did not reveal an appreciable change of 15 frameshift efficiency, we carried out the same experiments in vitro using RRL to monitor the effects in a single mRNA setup. Replacement of the entire nascent chain with an unrelated sequence leads to a 35% increase in frameshifting (Fig. 4F) . The extent of this change is clearly not due to loss of the 5' attenuator loop, as indicated by an experiment where silent attenuator loop mutations result in only a slight increase in frameshifting. Mutation of the leucine 4386 and 20 arginine 4387 to alanine led to a considerable (30%) increase in frameshifting (Fig. 4F-G) , implying that these nascent chain interactions with the ribosomal exit tunnel play an important role in regulating frameshifting levels, possibly mechanistically akin to the well-studied SecM stalling system in bacteria (33) , where it was shown that co-translational folding and the translocon-induced mechanical force can rescue the stall induced by interactions between the 25 nascent chain and the ribosomal tunnel (34). These observations also imply that any cellular nascent-chain factors (35, 36) , or even the intracellular concentration of Zn 2+ , which is known to change in response to viral infections (37) , might influence the rate of frameshifting.

Our results provide a mechanistic description of frameshifting that occurs during translation of the SARS-CoV-2 genome and reveal the features that may be exploited by the virus to finely control the stoichiometry of viral proteins at different stages of infection (Fig. 5) . Interfering with 5 the frameshifting process at the level of nascent chain interactions with the ribosomal tunnel, at the level of RNA folding that leads to the formation of the frameshift stimulatory pseudoknot, or to perturb the interactions between the pseudoknot and the mRNA channel, represent a viable strategy in our search for new drugs against SARS-CoV-2, the virus that is currently causing the global COVID-19 pandemic. Our results will also be useful for understanding the mechanism of 10 programmed ribosomal "-1" frameshifting employed by many other medically important viruses.

We thank A. Jomaa for advice on cryo-EM data processing and A. Picenoni for help with the grid preparation. We are indebted to the ETH scientific center for optical and electron microscopy (ScopeM) for access to electron microscopes, and in particular to M. 15 Peterek and D. Boehringer. 30 Electron Microscopy Data Bank as EMD-YYYY, respectively. The structure refined into the further classified particle set reconstruction and the corresponding maps are available as PDB-ZZZZ and EMD-WWWW, respectively.

Figures S1-S5

Tables S1-S2 The observed interactions between the pseudoknot and the ribosome prime the system for frameshifting. The features of the pseudoknot and the interactions between the nascent chain and 5 the ribosomal tunnel play a role in the efficiency of frameshifting. The efficiency of frameshifting is increased by the presence of a stop codon near the frameshifting site. Ribosomes that progress beyond the frameshifting site in the 0 frame quickly terminate and disassemble, thereby increasing the chances that the pseudoknot will refold before it is encountered by the closely trailing ribosome. The trailing ribosome in turn encounters the pseudoknot, which 10 increases the possibility of undergoing -1 frameshifting.

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