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1.
Nat Commun ; 12(1): 5695, 2021 09 28.
Article in English | MEDLINE | ID: covidwho-1442780

ABSTRACT

The dynamics of SARS-CoV-2 RNA structure and their functional relevance are largely unknown. Here we develop a simplified SPLASH assay and comprehensively map the in vivo RNA-RNA interactome of SARS-CoV-2 genome across viral life cycle. We report canonical and alternative structures including 5'-UTR and 3'-UTR, frameshifting element (FSE) pseudoknot and genome cyclization in both cells and virions. We provide direct evidence of interactions between Transcription Regulating Sequences, which facilitate discontinuous transcription. In addition, we reveal alternative short and long distance arches around FSE. More importantly, we find that within virions, while SARS-CoV-2 genome RNA undergoes intensive compaction, genome domains remain stable but with strengthened demarcation of local domains and weakened global cyclization. Taken together, our analysis reveals the structural basis for the regulation of replication, discontinuous transcription and translational frameshifting, the alternative conformations and the maintenance of global genome organization during the whole life cycle of SARS-CoV-2, which we anticipate will help develop better antiviral strategies.


Subject(s)
Frameshifting, Ribosomal/genetics , Genome, Viral/genetics , RNA, Viral/genetics , SARS-CoV-2/genetics , Animals , COVID-19/virology , Chlorocebus aethiops , Humans , RNA-Seq , Transcription, Genetic , Vero Cells , Virus Replication/genetics
2.
Viruses ; 13(7)2021 06 25.
Article in English | MEDLINE | ID: covidwho-1389549

ABSTRACT

The product of the interferon-stimulated gene C19orf66, Shiftless (SHFL), restricts human immunodeficiency virus replication through downregulation of the efficiency of the viral gag/pol frameshifting signal. In this study, we demonstrate that bacterially expressed, purified SHFL can decrease the efficiency of programmed ribosomal frameshifting in vitro at a variety of sites, including the RNA pseudoknot-dependent signals of the coronaviruses IBV, SARS-CoV and SARS-CoV-2, and the protein-dependent stimulators of the cardioviruses EMCV and TMEV. SHFL also reduced the efficiency of stop-codon readthrough at the murine leukemia virus gag/pol signal. Using size-exclusion chromatography, we confirm the binding of the purified protein to mammalian ribosomes in vitro. Finally, through electrophoretic mobility shift assays and mutational analysis, we show that expressed SHFL has strong RNA binding activity that is necessary for full activity in the inhibition of frameshifting, but shows no clear specificity for stimulatory RNA structures.


Subject(s)
Codon, Terminator/genetics , Coronavirus/genetics , Frameshifting, Ribosomal/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Viral Proteins/metabolism , Base Sequence , Escherichia coli/genetics , Gene Expression Regulation, Viral , Humans , Leukemia Virus, Murine/genetics , RNA Recognition Motif Proteins , RNA, Viral/genetics , Virus Replication
3.
Nat Commun ; 12(1): 4749, 2021 08 06.
Article in English | MEDLINE | ID: covidwho-1345559

ABSTRACT

The RNA pseudoknot that stimulates programmed ribosomal frameshifting in SARS-CoV-2 is a possible drug target. To understand how it responds to mechanical tension applied by ribosomes, thought to play a key role during frameshifting, we probe its structural dynamics using optical tweezers. We find that it forms multiple structures: two pseudoknotted conformers with different stability and barriers, and alternative stem-loop structures. The pseudoknotted conformers have distinct topologies, one threading the 5' end through a 3-helix junction to create a knot-like fold, the other with unthreaded 5' end, consistent with structures observed via cryo-EM and simulations. Refolding of the pseudoknotted conformers starts with stem 1, followed by stem 3 and lastly stem 2; Mg2+ ions are not required, but increase pseudoknot mechanical rigidity and favor formation of the knot-like conformer. These results resolve the SARS-CoV-2 frameshift signal folding mechanism and highlight its conformational heterogeneity, with important implications for structure-based drug-discovery efforts.


Subject(s)
Frameshifting, Ribosomal/genetics , Nucleic Acid Conformation , RNA, Viral/genetics , Ribosomes/physiology , SARS-CoV-2/genetics , COVID-19 , Frameshift Mutation/genetics , Humans , Optical Tweezers , RNA, Messenger/genetics
4.
Proc Natl Acad Sci U S A ; 118(26)2021 06 29.
Article in English | MEDLINE | ID: covidwho-1284758

ABSTRACT

Translation of open reading frame 1b (ORF1b) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires a programmed -1 ribosomal frameshift (-1 PRF) promoted by an RNA pseudoknot. The extent to which SARS-CoV-2 replication may be sensitive to changes in -1 PRF efficiency is currently unknown. Through an unbiased, reporter-based high-throughput compound screen, we identified merafloxacin, a fluoroquinolone antibacterial, as a -1 PRF inhibitor for SARS-CoV-2. Frameshift inhibition by merafloxacin is robust to mutations within the pseudoknot region and is similarly effective on -1 PRF of other betacoronaviruses. Consistent with the essential role of -1 PRF in viral gene expression, merafloxacin impedes SARS-CoV-2 replication in Vero E6 cells, thereby providing proof-of-principle for targeting -1 PRF as a plausible and effective antiviral strategy for SARS-CoV-2 and other coronaviruses.


Subject(s)
Antiviral Agents/pharmacology , Frameshifting, Ribosomal/drug effects , SARS-CoV-2/drug effects , Virus Replication/drug effects , Animals , Betacoronavirus , Chlorocebus aethiops , Fluoroquinolones/pharmacology , Frameshifting, Ribosomal/genetics , Mutation , Nucleic Acid Conformation , RNA, Viral/chemistry , RNA, Viral/genetics , SARS-CoV-2/physiology , Vero Cells
5.
Nucleic Acids Res ; 49(6): 3092-3108, 2021 04 06.
Article in English | MEDLINE | ID: covidwho-1123330

ABSTRACT

The rapid spread of COVID-19 is motivating development of antivirals targeting conserved SARS-CoV-2 molecular machinery. The SARS-CoV-2 genome includes conserved RNA elements that offer potential small-molecule drug targets, but most of their 3D structures have not been experimentally characterized. Here, we provide a compilation of chemical mapping data from our and other labs, secondary structure models, and 3D model ensembles based on Rosetta's FARFAR2 algorithm for SARS-CoV-2 RNA regions including the individual stems SL1-8 in the extended 5' UTR; the reverse complement of the 5' UTR SL1-4; the frameshift stimulating element (FSE); and the extended pseudoknot, hypervariable region, and s2m of the 3' UTR. For eleven of these elements (the stems in SL1-8, reverse complement of SL1-4, FSE, s2m and 3' UTR pseudoknot), modeling convergence supports the accuracy of predicted low energy states; subsequent cryo-EM characterization of the FSE confirms modeling accuracy. To aid efforts to discover small molecule RNA binders guided by computational models, we provide a second set of similarly prepared models for RNA riboswitches that bind small molecules. Both datasets ('FARFAR2-SARS-CoV-2', https://github.com/DasLab/FARFAR2-SARS-CoV-2; and 'FARFAR2-Apo-Riboswitch', at https://github.com/DasLab/FARFAR2-Apo-Riboswitch') include up to 400 models for each RNA element, which may facilitate drug discovery approaches targeting dynamic ensembles of RNA molecules.


Subject(s)
Consensus , Models, Molecular , Nucleic Acid Conformation , RNA, Viral/chemistry , SARS-CoV-2/genetics , 3' Untranslated Regions/genetics , 5' Untranslated Regions/genetics , Algorithms , Aptamers, Nucleotide/genetics , Base Sequence , Binding Sites , Cryoelectron Microscopy , Datasets as Topic , Drug Evaluation, Preclinical/methods , Frameshifting, Ribosomal/genetics , Genome, Viral/genetics , RNA Stability , RNA, Viral/genetics , Reproducibility of Results , Riboswitch/genetics , Small Molecule Libraries/chemistry
6.
Biophys J ; 120(6): 1040-1053, 2021 03 16.
Article in English | MEDLINE | ID: covidwho-1083814

ABSTRACT

With the rapid rate of COVID-19 infections and deaths, treatments and cures besides hand washing, social distancing, masks, isolation, and quarantines are urgently needed. The treatments and vaccines rely on the basic biophysics of the complex viral apparatus. Although proteins are serving as main drug and vaccine targets, therapeutic approaches targeting the 30,000 nucleotide RNA viral genome form important complementary approaches. Indeed, the high conservation of the viral genome, its close evolutionary relationship to other viruses, and the rise of gene editing and RNA-based vaccines all argue for a focus on the RNA agent itself. One of the key steps in the viral replication cycle inside host cells is the ribosomal frameshifting required for translation of overlapping open reading frames. The RNA frameshifting element (FSE), one of three highly conserved regions of coronaviruses, is believed to include a pseudoknot considered essential for this ribosomal switching. In this work, we apply our graph-theory-based framework for representing RNA secondary structures, "RAG (or RNA-As-Graphs)," to alter key structural features of the FSE of the SARS-CoV-2 virus. Specifically, using RAG machinery of genetic algorithms for inverse folding adapted for RNA structures with pseudoknots, we computationally predict minimal mutations that destroy a structurally important stem and/or the pseudoknot of the FSE, potentially dismantling the virus against translation of the polyproteins. Our microsecond molecular dynamics simulations of mutant structures indicate relatively stable secondary structures. These findings not only advance our computational design of RNAs containing pseudoknots, they pinpoint key residues of the SARS-CoV-2 virus as targets for antiviral drugs and gene editing approaches.


Subject(s)
Frameshifting, Ribosomal/genetics , Mutation/genetics , RNA, Viral/chemistry , RNA, Viral/genetics , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Algorithms , Gene Editing , Molecular Dynamics Simulation , Nucleic Acid Conformation
7.
Biochem Soc Trans ; 49(1): 341-352, 2021 02 26.
Article in English | MEDLINE | ID: covidwho-997958

ABSTRACT

Coronaviruses (CoV) are positive-sense single-stranded RNA viruses, harboring the largest viral RNA genomes known to date. Apart from the primary sequence encoding for all the viral proteins needed for the generation of new viral particles, certain regions of CoV genomes are known to fold into stable structures, controlling several aspects of CoV life cycle, from the regulation of the discontinuous transcription of subgenomic mRNAs, to the packaging of the genome into new virions. Here we review the current knowledge on CoV RNA structures, discussing it in light of the most recent discoveries made possible by analyses of the SARS-CoV-2 genome.


Subject(s)
Coronavirus/genetics , Frameshifting, Ribosomal/genetics , Genome, Viral/genetics , RNA, Ribosomal/genetics , RNA, Viral/genetics , SARS-CoV-2/genetics , COVID-19/prevention & control , COVID-19/virology , Humans , Models, Molecular , Nucleic Acid Conformation , RNA, Ribosomal/chemistry , RNA, Viral/chemistry , SARS-CoV-2/physiology
8.
Virology ; 554: 75-82, 2021 02.
Article in English | MEDLINE | ID: covidwho-989370

ABSTRACT

Human population growth, climate change, and globalization are accelerating the emergence of novel pathogenic viruses. In the past two decades alone, three such members of the coronavirus family have posed serious threats, spurring intense efforts to understand their biology as a way to identify targetable vulnerabilities. Coronaviruses use a programmed -1 ribosomal frameshift (-1 PRF) mechanism to direct synthesis of their replicase proteins. This is a critical switch in their replication program that can be therapeutically targeted. Here, we discuss how nearly half a century of research into -1 PRF have provided insight into the virological importance of -1 PRF, the molecular mechanisms that drive it, and approaches that can be used to manipulate it towards therapeutic outcomes with particular emphasis on SARS-CoV-2.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus/drug effects , Coronavirus/genetics , Frameshifting, Ribosomal/drug effects , Antiviral Agents/chemistry , Antiviral Agents/therapeutic use , Coronavirus/growth & development , Coronavirus/physiology , Coronavirus Infections/drug therapy , Frameshifting, Ribosomal/genetics , Frameshifting, Ribosomal/physiology , Gene Expression Regulation, Viral , Humans , Mutation , Nucleic Acid Conformation , RNA, Viral/chemistry , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , SARS-CoV-2/growth & development , SARS-CoV-2/physiology , Virus Replication
9.
Nucleic Acids Res ; 48(22): 12415-12435, 2020 12 16.
Article in English | MEDLINE | ID: covidwho-917705

ABSTRACT

The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5' end, the ribosomal frameshift segment and the 3'-untranslated region (3'-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.


Subject(s)
COVID-19/prevention & control , Magnetic Resonance Spectroscopy/methods , Nucleic Acid Conformation , RNA, Viral/chemistry , SARS-CoV-2/genetics , 3' Untranslated Regions/genetics , Base Sequence , COVID-19/epidemiology , COVID-19/virology , Frameshifting, Ribosomal/genetics , Genome, Viral/genetics , Humans , Models, Molecular , Pandemics , SARS-CoV-2/physiology
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