Structural and functional conservation of the programmed −1 ribosomal frameshift signal of SARS coronavirus 2 (SARS-CoV-2)

Abstract

Approximately 17 years after the severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic, the world is currently facing the COVID-19 pandemic caused by SARS corona virus 2 (SARS-CoV-2). According to the most optimistic projections, it will take more than a year to develop a vaccine, so the best short-term strategy may lie in identifying virus-specific targets for small molecule–based interventions. All coronaviruses utilize a molecular mechanism called programmed −1 ribosomal frameshift (−1 PRF) to control the relative expression of their proteins. Previous analyses of SARS-CoV have revealed that it employs a structurally unique three-stemmed mRNA pseudoknot that stimulates high −1 PRF rates and that it also harbors a −1 PRF attenuation element. Altering −1 PRF activity impairs virus replication, suggesting that this activity may be therapeutically targeted. Here, we comparatively analyzed the SARS-CoV and SARS-CoV-2 frameshift signals. Structural and functional analyses revealed that both elements promote similar −1 PRF rates and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablate −1 PRF activity. We noted that the upstream attenuator hairpin activity is also functionally retained in both viruses, despite differences in the primary sequence in this region. Small-angle X-ray scattering analyses indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 have the same conformation. Finally, a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit −1 PRF was similarly effective against −1 PRF in SARS-CoV-2, suggesting that such frameshift inhibitors may be promising lead compounds to combat the current COVID-19 pandemic. Approximately 17 years after the severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic, the world is currently facing the COVID-19 pandemic caused by SARS corona virus 2 (SARS-CoV-2). According to the most optimistic projections, it will take more than a year to develop a vaccine, so the best short-term strategy may lie in identifying virus-specific targets for small molecule–based interventions. All coronaviruses utilize a molecular mechanism called programmed −1 ribosomal frameshift (−1 PRF) to control the relative expression of their proteins. Previous analyses of SARS-CoV have revealed that it employs a structurally unique three-stemmed mRNA pseudoknot that stimulates high −1 PRF rates and that it also harbors a −1 PRF attenuation element. Altering −1 PRF activity impairs virus replication, suggesting that this activity may be therapeutically targeted. Here, we comparatively analyzed the SARS-CoV and SARS-CoV-2 frameshift signals. Structural and functional analyses revealed that both elements promote similar −1 PRF rates and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablate −1 PRF activity. We noted that the upstream attenuator hairpin activity is also functionally retained in both viruses, despite differences in the primary sequence in this region. Small-angle X-ray scattering analyses indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 have the same conformation. Finally, a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit −1 PRF was similarly effective against −1 PRF in SARS-CoV-2, suggesting that such frameshift inhibitors may be promising lead compounds to combat the current COVID-19 pandemic. SARS-CoV-2, the etiological agent of COVID-19, is a member of the coronavirus family (1Coronaviridae Study Group of the International Committee on Taxonomy of Viruses The species severe acute respiratory syndrome–related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2.Nat. Microbiol. 2020; 5 (32123347): 536-54410.1038/s41564-020-0695-zCrossref PubMed Scopus (4266) Google Scholar). Coronaviruses have (+) single-strand RNA genomes that harbor two long ORFs that occupy approximately two-thirds of the 5′ end of the genomic RNA (ORF1 and ORF2), followed by several ORFs that are expressed late in the viral replication cycle from subgenomic RNAs (Fig. 1A) (2Brian D.A. Baric R.S. Coronavirus genome structure and replication.Coronavirus Replication and Reverse Genetics. Springer-Verlag, Berlin/Heidelberg2005: 1-30Crossref Scopus (467) Google Scholar). In general, the immediate early proteins encoded by ORF1a are involved in ablating the host cellular innate immune response, whereas the early proteins encoded in ORF1b are involved in genome replication and RNA synthesis. These functions include generating the minus-strand replicative intermediate, new plus-strand genomic RNAs, and subgenomic RNAs, which mostly encode structural, late proteins. ORF1b is out of frame with respect to ORF1a, and all coronaviruses utilize a molecular mechanism called programmed −1 ribosomal frameshifting (−1 PRF) as a means to synthesize the ORF2-encoded proteins (3Dinman J.D. Mechanisms and implications of programmed translational frameshifting.Wiley Interdiscip. Rev. RNA. 2012; 3 (22715123): 661-67310.1002/wrna.1126Crossref PubMed Scopus (138) Google Scholar, 4Atkins J.F. Loughran G. Bhatt P.R. Firth A.E. Baranov P.V. Ribosomal frameshifting and transcriptional slippage: From genetic steganography and cryptography to adventitious use.Nucleic Acids Res. 2016; 44 (27436286): 7007-707810.1093/nar/gkw530PubMed Google Scholar). −1 PRF is a mechanism in which cis-acting elements in the mRNA direct elongating ribosomes to shift the reading frame by 1 base in the 5′ direction. The use of a −1 PRF mechanism for expression of a viral gene was first identified in the Rous sarcoma virus (5Jacks T. Varmus H.E. Expression of the Rous sarcoma virus pol gene by ribosomal frameshifting.Science. 1985; 230 (2416054): 1237-124210.1126/science.2416054Crossref PubMed Scopus (287) Google Scholar). A −1 PRF mechanism was shown to be required to translate ORF1ab in a coronavirus, avian infectious bronchitis virus, 2 years later (6Brierley I. Boursnell M.E. Binns M.M. Bilimoria B. Blok V.C. Brown T.D. Inglis S.C. An efficient ribosomal frame-shifting signal in the polymerase-encoding region of the coronavirus IBV.EMBO J. 1987; 6 (3428275): 3779-378510.1002/j.1460-2075.1987.tb02713.xCrossref PubMed Scopus (227) Google Scholar). In coronaviruses, −1 PRF functions as a developmental switch, and mutations and small molecules that alter this process have deleterious effects on virus replication (7Plant E.P.P. Rakauskaite R. Taylor D.R.R. Dinman J.D.D. Achieving a golden mean: mechanisms by which coronaviruses ensure synthesis of the correct stoichiometric ratios of viral proteins.J.Virol. 2010; 84 (20164235): 4330-434010.1128/JVI.02480-09Crossref PubMed Scopus (81) Google Scholar, 8Plant E. Sims A. Baric R. Dinman J. Taylor D. Altering SARS coronavirus frameshift efficiency affects genomic and subgenomic RNA production.Viruses. 2013; 5 (23334702): 279-29410.3390/v5010279Crossref PubMed Scopus (39) Google Scholar). The −1 PRF signal can be broken down into three discrete parts: the “slippery site,” a linker region, and a downstream stimulatory region of mRNA secondary structure, typically an mRNA pseudoknot (reviewed in Ref. 3Dinman J.D. Mechanisms and implications of programmed translational frameshifting.Wiley Interdiscip. Rev. RNA. 2012; 3 (22715123): 661-67310.1002/wrna.1126Crossref PubMed Scopus (138) Google Scholar). The primary sequence of the slippery site and its placement in relation to the incoming translational reading frame is critical: it must be N NNW WWZ (codons are shown in the incoming or 0-frame), where NNN is a stretch of three identical nucleotides, WWW is either AAA or UUU, and Z ≠ G. The linker region is less well-defined, but typically is short (1–12 nt long) and is thought to be important for determining the extent of −1 PRF in a virus-specific manner. The function of the downstream secondary structure is to induce elongating ribosomes to pause, a critical step for efficient −1 PRF to occur (reviewed in Ref. 9Rodnina M.V. Korniy N. Klimova M. Karki P. Peng B.-Z. Senyushkina T. Belardinelli R. Maracci C. Wohlgemuth I. Samatova E. Peske F. Translational recoding: canonical translation mechanisms reinterpreted.Nucleic Acids Res. 2020; 48 (31511883): 1056-106710.1093/nar/gkz783Crossref PubMed Scopus (10) Google Scholar). The generally accepted mechanism of −1 PRF is that the mRNA secondary structure directs elongating ribosomes to pause with its A- and P-site bound aminoacyl- and peptidyl-tRNAs positioned over the slippery site. The sequence of the slippery site allows for re-pairing of the tRNAs to the −1 frame codons after they “simultaneously slip” by 1 base in the 5′ direction along the mRNA. The subsequent resolution of the downstream mRNA secondary structure allows the ribosome to continue elongation of the nascent polypeptide in the new translational reading frame. The downstream stimulatory elements are most commonly H-type mRNA pseudoknots, so called because they are composed of two co-axially stacked stem loops where the second stem is formed by base pairing between sequence in the loop of the first-stem loop and additional downstream sequence (10Puglisi J.D. Wyatt J.R. Tinoco Jr., I. A pseudoknotted RNA oligonucleotide.Nature. 1988; 331 (3336440): 283-28610.1038/331283a0Crossref PubMed Scopus (75) Google Scholar). The SARS-CoV pseudoknot is more complex because it contains a third, internal stem-loop element (11Plant E.P. Pérez-Alvarado G.C. Jacobs J.L. Mukhopadhyay B. Hennig M. Dinman J.D. A three-stemmed mRNA pseudoknot in the SARS coronavirus frameshift signal.PLoS Biol. 2005; 3 (15884978): e17210.1371/journal.pbio.0030172Crossref PubMed Scopus (115) Google Scholar, 12Brierley I. Dos Ramos F.J. Programmed ribosomal frameshifting in HIV-1 and the SARS-CoV.Virus Res. 2006; 119 (16310880): 29-4210.1016/j.virusres.2005.10.008Crossref PubMed Scopus (113) Google Scholar, 13Baranov P.V. Henderson C.M. Anderson C.B. Gesteland R.F. Atkins J.F. Howard M.T. Programmed ribosomal frameshifting in decoding the SARS-CoV genome.Virology. 2005; 332 (15680415): 498-51010.1016/j.virol.2004.11.038Crossref PubMed Scopus (157) Google Scholar). Mutations affecting this structure decreased the rates of −1 PRF and had deleterious effects on virus propagation, thus suggesting that it may present a target for small-molecule therapeutics (7Plant E.P.P. Rakauskaite R. Taylor D.R.R. Dinman J.D.D. Achieving a golden mean: mechanisms by which coronaviruses ensure synthesis of the correct stoichiometric ratios of viral proteins.J.Virol. 2010; 84 (20164235): 4330-434010.1128/JVI.02480-09Crossref PubMed Scopus (81) Google Scholar, 8Plant E. Sims A. Baric R. Dinman J. Taylor D. Altering SARS coronavirus frameshift efficiency affects genomic and subgenomic RNA production.Viruses. 2013; 5 (23334702): 279-29410.3390/v5010279Crossref PubMed Scopus (39) Google Scholar). In addition, the presence of a hairpin located immediately 5′ of the slippery site has been reported to regulate −1 PRF by attenuating its activity (14Cho C.-P. Lin S.-C. Chou M.-Y. Hsu H.-T. Chang K.-Y. Regulation of programmed ribosomal frameshifting by co-translational refolding RNA hairpins.PLoS One. 2013; 8 (23638024): e6228310.1371/journal.pone.0062283Crossref PubMed Scopus (28) Google Scholar). Here, we report on the −1 PRF signal from SARS-CoV-2. The core −1 PRF signal is nearly identical to that of SARS-CoV, containing only a single-nucleotide difference, a C to A. This change maps to a loop region in the molecule that is not predicted to affect the structure of the three-stemmed pseudoknot. The primary sequence of the attenuator hairpin is less well-conserved. However, genetic analyses reveal that both elements appear to have been functionally conserved. Conservation of RNA structure is further supported by the similarity of the small-angle X-ray scattering profiles for the two pseudoknots and by the similar anti-frameshifting activity of a small-molecule ligand against both frameshift signals. The core of the SARS-CoV −1 PRF signal begins with the U UUA AAC slippery site, followed by a 6-nt spacer region and then the three-stemmed mRNA pseudoknot that stimulates −1 PRF. A second regulatory element, called the attenuator hairpin, is located 5′ of the slippery site. Pairwise analysis of the SARS-CoV and SARS-CoV-2 frameshift signals revealed that the sequence of the attenuator hairpin was less well-conserved than the frameshift-stimulating pseudoknot (Fig. 1B). The structure of the SARS-CoV −1 PRF signal was previously determined to include a three-stemmed pseudoknot (11Plant E.P. Pérez-Alvarado G.C. Jacobs J.L. Mukhopadhyay B. Hennig M. Dinman J.D. A three-stemmed mRNA pseudoknot in the SARS coronavirus frameshift signal.PLoS Biol. 2005; 3 (15884978): e17210.1371/journal.pbio.0030172Crossref PubMed Scopus (115) Google Scholar). Using this structure as a guide, the single C-to-A base difference between the core SARS-CoV and SARS-CoV-2 −1 PRF signals (Fig. 1B) that maps to a loop that is not predicted to alter the structure of the −1 PRF stimulating element (7Plant E.P.P. Rakauskaite R. Taylor D.R.R. Dinman J.D.D. Achieving a golden mean: mechanisms by which coronaviruses ensure synthesis of the correct stoichiometric ratios of viral proteins.J.Virol. 2010; 84 (20164235): 4330-434010.1128/JVI.02480-09Crossref PubMed Scopus (81) Google Scholar) (Fig. 1C). In contrast, the attenuator hairpin contains six differences in the nucleotide sequence between the two viruses (Fig. 1B), and the SARS-CoV-2 element is predicted to be less stable than its SARS-CoV counterpart (Fig. 1D). To determine the importance of each of these elements, a series of silent coding mutants of both the SARS-CoV and SARS-CoV-2 sequences were constructed to disrupt the putative attenuators, slippery sites, and stems 1, 2, and 3 of the pseudoknots (Fig. 1, E and F). Standard Dual-Luciferase assays were used to monitor −1 PRF activities of the two −1 PRF signals (15Harger J.W. Dinman J.D. An in vivo Dual-Luciferase assay system for studying translational recoding in the yeast Saccharomyces cerevisiae.RNA. 2003; 9 (12869712): 1019-102410.1261/rna.5930803Crossref PubMed Scopus (120) Google Scholar, 16Jacobs J.L. Dinman J.D. Systematic analysis of bicistronic reporter assay data.Nucleic Acids Res. 2004; 32 (15561995): e16010.1093/nar/gnh157Crossref PubMed Scopus (77) Google Scholar) in cultured human cell lines. For both of the elements, −1 PRF activity was ∼20% in HEK (Fig. 2A) and ∼30% in HeLa (Fig. 2B). Amino acid sequence silent coding mutation of the U UUA AAC slippery sites to C CUC AAC (the incoming 0-frame codons are indicated by spaces) ablated −1 PRF activity in both cases to less than 1% (Fig. 2, A and B), demonstrating the functional conservation of this central feature of the −1 PRF signal. To test functional conservation of the three-stemmed pseudoknot, a series of silent 0-frame coding mutations were made to each of the stems in both the SARS-CoV and SARS-CoV-2 frameshift signals, and assays were performed in HEK cells. Disruption of stem 1 strongly suppressed the ability of both elements to promote −1 PRF, decreasing rates to 0.67 ± 0.03 and 0.7 ± 0.1% for SARS-CoV and SARS-CoV-2, respectively, p < 0.0001 (Fig. 2C). Similarly, disruption of stem 2 had a strong negative impact on −1 PRF, decreasing rates to 0.68 ± 0.04% for SARS-CoV and 0.8 ± 0.1% for SARS-CoV-2; p < 0.0001 (Fig. 2D). In contrast, although disruption of stem 3 did decrease −1 PRF efficiencies, the effects were less severe, although the decreases were statistically significant (13.1 ± 0.9 and 8 ± 1% for SARS-CoV and SARS-CoV-2, respectively; p < 0.0001) (Fig. 2E). These the that the structure and function of the core −1 PRF signals have been between the two the presence of an element located immediately 5′ of the SARS-CoV slippery site that had the ability to decrease −1 PRF, called the attenuator hairpin (14Cho C.-P. Lin S.-C. Chou M.-Y. Hsu H.-T. Chang K.-Y. Regulation of programmed ribosomal frameshifting by co-translational refolding RNA hairpins.PLoS One. 2013; 8 (23638024): e6228310.1371/journal.pone.0062283Crossref PubMed Scopus (28) Google Scholar). less well-conserved the primary sequence (Fig. 1, and of this sequence into the SARS-CoV-2 reporter also in decreased −1 PRF ± with ± the attenuator hairpin < whereas disruption of the hairpin did not in decreased efficiency ± p (Fig. In the control the SARS-CoV attenuator also decreased −1 PRF, to a extent ± with ± the attenuator hairpin and ± 1% with the hairpin (Fig. the attenuation function has also been between the two viruses despite the differences in primary nucleotide on the strong conservation of the frameshift signal between SARS-CoV and SARS-CoV-2, we a frameshift against the first also retained activity against the We on a small-molecule ligand previously shown to bind to the SARS-CoV pseudoknot and −1 PRF, acid as of RNA ligand that the −1 ribosomal frameshifting of by PubMed Scopus Google Scholar, J. M.T. ligand the of the SARS virus PubMed Scopus Google Scholar). the −1 PRF activity from Dual-Luciferase in in the presence and of we that 5 −1 PRF activity by from ± 3 to ± 1% (Fig. This was but that previously for the SARS-CoV pseudoknot, where 0.8 −1 PRF by of RNA ligand that the −1 ribosomal frameshifting of by PubMed Scopus Google Scholar). Finally, we used small and X-ray scattering to the scattering profiles of the two pseudoknots, which their The scattering profiles as a function of the scattering were for of SARS-CoV (Fig. and SARS-CoV-2 (Fig. The difference between their scattering profiles is with all (Fig. The of the is to the molecular of the structure of RNA and B. PubMed Scopus Google the similarity of the profiles for the two pseudoknots that their are the SARS-CoV pseudoknots can D. E.P. Sims Pérez-Alvarado G.C. Baric R.S. Dinman J.D. Taylor Hennig M. Hennig M. RNA a in ribosomal frameshifting of the SARS Acids Res. 2013; PubMed Scopus Google we also performed where the RNA was by immediately X-ray to ensure only were From profiles (Fig. we determined the as the of We the same for SARS-CoV and SARS-CoV-2 ± and ± The difference for this is also with for all (Fig. These that SARS-CoV-2 have a functional −1 PRF site. also that the of the frameshift signal in SARS-CoV-2 are similar to of the frameshift signal in only was the of −1 PRF to identical for both viruses, but stems 1 and 2 in the stimulatory pseudoknot frameshifting in both whereas stem 3 −1 PRF but did not it in each each frameshift signal an attenuator hairpin that decreased −1 PRF and the of two pseudoknots as in the scattering profiles were The in the of the pseudoknots from SARS-CoV and SARS-CoV-2 that of the that have been in are to over to SARS-CoV-2. For of stem 3 will lead to or change in −1 PRF, whereas mutation of the A in stem 2 will −1 PRF (7Plant E.P.P. Rakauskaite R. Taylor D.R.R. Dinman J.D.D. Achieving a golden mean: mechanisms by which coronaviruses ensure synthesis of the correct stoichiometric ratios of viral proteins.J.Virol. 2010; 84 (20164235): 4330-434010.1128/JVI.02480-09Crossref PubMed Scopus (81) Google Scholar, 8Plant E. Sims A. Baric R. Dinman J. Taylor D. Altering SARS coronavirus frameshift efficiency affects genomic and subgenomic RNA production.Viruses. 2013; 5 (23334702): 279-29410.3390/v5010279Crossref PubMed Scopus (39) Google the pseudoknot will between loop 2 D. E.P. Sims Pérez-Alvarado G.C. Baric R.S. Dinman J.D. Taylor Hennig M. Hennig M. RNA a in ribosomal frameshifting of the SARS Acids Res. 2013; PubMed Scopus Google and of −1 PRF will most viral (11Plant E.P. Pérez-Alvarado G.C. Jacobs J.L. Mukhopadhyay B. Hennig M. Dinman J.D. A three-stemmed mRNA pseudoknot in the SARS coronavirus frameshift signal.PLoS Biol. 2005; 3 (15884978): e17210.1371/journal.pbio.0030172Crossref PubMed Scopus (115) Google Scholar). This of SARS-CoV-2 to attenuation by −1 PRF is of because it that −1 PRF may a promising for Previous on SARS-CoV that inhibit both −1 PRF and virus replication E.P. F. Taylor J.W. of ribosomal frameshifting by SARS coronavirus Res. PubMed Scopus Google Scholar). The that the which was in a for −1 PRF inhibitors in SARS-CoV of RNA ligand that the −1 ribosomal frameshifting of by PubMed Scopus Google is similarly −1 PRF in SARS-CoV-2 for small-molecule frameshifting inhibitors in SARS-CoV-2 and the that the pseudoknot may be an The SARS-CoV-2 −1 PRF signal was identified from the genome sequence sequence The was used to sequences in the SARS-CoV-2 genome most similar to the SARS-CoV −1 PRF was reported between and of SARS-CoV-2 that was identical to the SARS The SARS-CoV-2 sequence contains a single mutation from C to A base was used to between sequences from SARS-CoV nt end nt and SARS-CoV-2 nt end nt for Dual-Luciferase assays for SARS-CoV-2 were by of the reporter E. Sims A. Baric R. Dinman J. Taylor D. Altering SARS coronavirus frameshift efficiency affects genomic and subgenomic RNA production.Viruses. 2013; 5 (23334702): 279-29410.3390/v5010279Crossref PubMed Scopus (39) Google a single C-to-A mutation base to the mutation in the SARS-CoV-2 genome were and by were into and containing were by The frameshift reporter negative and reporter containing silent mutations the −1 PRF slippery site stem 1 stem 3 and attenuator were constructed similarly by containing silent mutations to stem 2 were made by with and and a containing the silent mutations to stem 2 of SARS and SARS-CoV-2 into the for Dual-Luciferase assays were made as previously M.T. in a frameshift stimulatory PubMed Scopus Google Scholar). the reporter was made by the sequence for and SARS-CoV-2 frameshift signal in the frame upstream of the sequence in the with in the −1 frame. A negative control was made by of the slippery sequence with a and a control was made a frameshift signal and the two RNA were from these by and in by RNA for for were by an containing the sequences of the SARS-CoV and SARS-CoV-2 pseudoknots into the and sites of the as previously D.A. M.T. Programmed −1 frameshifting efficiency with RNA pseudoknot not to 2012; PubMed Scopus Google Scholar). RNA were from these by three upstream of the pseudoknot and downstream The was on its 5′ end to include the The were then in by RNA used in this are shown in and HeLa were from the were in with 1% 1% 1% and in HeLa were in with 1% and in and HeLa were into The were after with of Dual-Luciferase reporter the The frameshifting efficiency of the reporter in cultured was as previously (15Harger J.W. Dinman J.D. An in vivo Dual-Luciferase assay system for studying translational recoding in the yeast Saccharomyces cerevisiae.RNA. 2003; 9 (12869712): 1019-102410.1261/rna.5930803Crossref PubMed Scopus (120) Google Scholar, 16Jacobs J.L. Dinman J.D. Systematic analysis of bicistronic reporter assay data.Nucleic Acids Res. 2004; 32 (15561995): e16010.1093/nar/gnh157Crossref PubMed Scopus (77) Google Scholar) a Dual-Luciferase reporter assay system after the were with and then with were in in a and activity was a The frameshift was by the three or and then a of to was with a control to The of ratios for each is the frameshift for the A of three were for each each of which were in of each are on with of the for analyses were test or analysis of as 8 To −1 PRF efficiency in 2 of mRNA from each was to with of and of 1 acid and and then for The from each of the was after of each with of for and then was after of and The −1 PRF efficiency was from the of and the from the negative control and by the were made and were made with RNA for were made by in of followed by of the RNA. 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