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1.
J Vis Exp ; (185)2022 Jul 12.
Article in English | MEDLINE | ID: covidwho-1964147

ABSTRACT

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved automated acquisition strategies, preparative techniques that expand the possibilities of what the electron microscope can image at high-resolution using cryo-ET and new subtomogram averaging software. Additionally, data acquisition has become increasingly streamlined, making it more accessible to many users. The SARS-CoV-2 pandemic has further accelerated remote cryo-electron microscopy (cryo-EM) data collection, especially for single-particle cryo-EM, in many facilities globally, providing uninterrupted user access to state-of-the-art instruments during the pandemic. With the recent advances in Tomo5 (software for 3D electron tomography), remote cryo-ET data collection has become robust and easy to handle from anywhere in the world. This article aims to provide a detailed walk-through, starting from the data collection setup in the tomography software for the process of a (remote) cryo-ET data collection session with detailed troubleshooting. The (remote) data collection protocol is further complemented with the workflow for structure determination at near-atomic resolution by subtomogram averaging with emClarity, using apoferritin as an example.


Subject(s)
COVID-19 , Electron Microscope Tomography , Cryoelectron Microscopy/methods , Data Collection , Electron Microscope Tomography/methods , Humans , Image Processing, Computer-Assisted/methods , SARS-CoV-2
2.
Acta Crystallogr D Struct Biol ; 78(Pt 7): 806-816, 2022 Jul 01.
Article in English | MEDLINE | ID: covidwho-1922451

ABSTRACT

The availability of new artificial intelligence-based protein-structure-prediction tools has radically changed the way that cryo-EM maps are interpreted, but it has not eliminated the challenges of map interpretation faced by a microscopist. Models will continue to be locally rebuilt and refined using interactive tools. This inevitably results in occasional errors, among which register shifts remain one of the most difficult to identify and correct. Here, checkMySequence, a fast, fully automated and parameter-free method for detecting register shifts in protein models built into cryo-EM maps, is introduced. It is shown that the method can assist model building in cases where poorer map resolution hinders visual interpretation. It is also shown that checkMySequence could have helped to avoid a widely discussed sequence-register error in a model of SARS-CoV-2 RNA-dependent RNA polymerase that was originally detected thanks to a visual residue-by-residue inspection by members of the structural biology community. The software is freely available at https://gitlab.com/gchojnowski/checkmysequence.


Subject(s)
Artificial Intelligence , COVID-19 , Cryoelectron Microscopy/methods , Humans , Models, Molecular , Proteins/chemistry , RNA, Viral , SARS-CoV-2
3.
Viruses ; 14(2)2022 02 17.
Article in English | MEDLINE | ID: covidwho-1786043

ABSTRACT

Various adenoviruses are being used as viral vectors for the generation of vaccines against chronic and emerging diseases (e.g., AIDS, COVID-19). Here, we report the improved capsid structure for one of these vectors, human adenovirus D26 (HAdV-D26), at 3.4 Å resolution, by reprocessing the previous cryo-electron microscopy dataset and obtaining a refined model. In addition to overall improvements in the model, the highlights of the structure include (1) locating a segment of the processed peptide of VIII that was previously believed to be released from the mature virions, (2) reorientation of the helical appendage domain (APD) of IIIa situated underneath the vertex region relative to its counterpart observed in the cleavage defective (ts1) mutant of HAdV-C5 that resulted in the loss of interactions between the APD and hexon bases, and (3) the revised conformation of the cleaved N-terminal segments of pre-protein VI (pVIn), located in the hexon cavities, is highly conserved, with notable stacking interactions between the conserved His13 and Phe18 residues. Taken together, the improved model of HAdV-D26 capsid provides a better understanding of protein-protein interactions in HAdV capsids and facilitates the efforts to modify and/or design adenoviral vectors with altered properties. Last but not least, we provide some insights into clotting factors (e.g., FX and PF4) binding to AdV vectors.


Subject(s)
Adenoviruses, Human/chemistry , Capsid/chemistry , Capsid/ultrastructure , Cryoelectron Microscopy/methods , Adenoviruses, Human/genetics , Capsid Proteins/genetics , Humans , Models, Molecular , Protein Conformation , Protein Interaction Domains and Motifs , Virus Assembly , Virus Internalization
4.
Annu Rev Biochem ; 91: 1-32, 2022 06 21.
Article in English | MEDLINE | ID: covidwho-1759478

ABSTRACT

Cryo-electron microscopy (cryo-EM) continues its remarkable growth as a method for visualizing biological objects, which has been driven by advances across the entire pipeline. Developments in both single-particle analysis and in situ tomography have enabled more structures to be imaged and determined to better resolutions, at faster speeds, and with more scientists having improved access. This review highlights recent advances at each stageof the cryo-EM pipeline and provides examples of how these techniques have been used to investigate real-world problems, including antibody development against the SARS-CoV-2 spike during the recent COVID-19 pandemic.


Subject(s)
COVID-19 , Pandemics , Cryoelectron Microscopy/methods , Humans , SARS-CoV-2 , Single Molecule Imaging
5.
Acta Crystallogr D Struct Biol ; 78(Pt 2): 152-161, 2022 Feb 01.
Article in English | MEDLINE | ID: covidwho-1684950

ABSTRACT

Recently, there has been a dramatic improvement in the quality and quantity of data derived using cryogenic electron microscopy (cryo-EM). This is also associated with a large increase in the number of atomic models built. Although the best resolutions that are achievable are improving, often the local resolution is variable, and a significant majority of data are still resolved at resolutions worse than 3 Å. Model building and refinement is often challenging at these resolutions, and hence atomic model validation becomes even more crucial to identify less reliable regions of the model. Here, a graphical user interface for atomic model validation, implemented in the CCP-EM software suite, is presented. It is aimed to develop this into a platform where users can access multiple complementary validation metrics that work across a range of resolutions and obtain a summary of evaluations. Based on the validation estimates from atomic models associated with cryo-EM structures from SARS-CoV-2, it was observed that models typically favor adopting the most common conformations over fitting the observations when compared with the model agreement with data. At low resolutions, the stereochemical quality may be favored over data fit, but care should be taken to ensure that the model agrees with the data in terms of resolvable features. It is demonstrated that further re-refinement can lead to improvement of the agreement with data without the loss of geometric quality. This also highlights the need for improved resolution-dependent weight optimization in model refinement and an effective test for overfitting that would help to guide the refinement process.


Subject(s)
Cryoelectron Microscopy/methods , Software Validation , Software , COVID-19 , Image Processing, Computer-Assisted , Models, Molecular , Reproducibility of Results , User-Computer Interface
6.
Nat Commun ; 13(1): 868, 2022 02 14.
Article in English | MEDLINE | ID: covidwho-1684025

ABSTRACT

SARS-CoV-2 infection is a major global public health concern with incompletely understood pathogenesis. The SARS-CoV-2 spike (S) glycoprotein comprises a highly conserved free fatty acid binding pocket (FABP) with unknown function and evolutionary selection advantage1,2. Deciphering FABP impact on COVID-19 progression is challenged by the heterogenous nature and large molecular variability of live virus. Here we create synthetic minimal virions (MiniVs) of wild-type and mutant SARS-CoV-2 with precise molecular composition and programmable complexity by bottom-up assembly. MiniV-based systematic assessment of S free fatty acid (FFA) binding reveals that FABP functions as an allosteric regulatory site enabling adaptation of SARS-CoV-2 immunogenicity to inflammation states via binding of pro-inflammatory FFAs. This is achieved by regulation of the S open-to-close equilibrium and the exposure of both, the receptor binding domain (RBD) and the SARS-CoV-2 RGD motif that is responsible for integrin co-receptor engagement. We find that the FDA-approved drugs vitamin K and dexamethasone modulate S-based cell binding in an FABP-like manner. In inflammatory FFA environments, neutralizing immunoglobulins from human convalescent COVID-19 donors lose neutralization activity. Empowered by our MiniV technology, we suggest a conserved mechanism by which SARS-CoV-2 dynamically couples its immunogenicity to the host immune response.


Subject(s)
COVID-19/immunology , Fatty Acids/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Virion/immunology , A549 Cells , Allosteric Site/genetics , Amino Acid Sequence , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Binding Sites/genetics , COVID-19/metabolism , COVID-19/virology , Cells, Cultured , Cryoelectron Microscopy/methods , Electron Microscope Tomography/methods , Fatty Acid-Binding Proteins/immunology , Fatty Acid-Binding Proteins/metabolism , Fatty Acids/metabolism , Humans , MCF-7 Cells , Microscopy, Confocal/methods , Protein Binding , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Sequence Homology, Amino Acid , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Virion/metabolism , Virion/ultrastructure
7.
Int J Mol Sci ; 22(24)2021 Dec 13.
Article in English | MEDLINE | ID: covidwho-1597050

ABSTRACT

Kininogens are multidomain glycoproteins found in the blood of most vertebrates. High molecular weight kininogen demonstrate both carrier and co-factor activity as part of the intrinsic pathway of coagulation, leading to thrombin generation. Kininogens are the source of the vasoactive nonapeptide bradykinin. To date, attempts to crystallize kininogen have failed, and very little is known about the shape of kininogen at an atomic level. New advancements in the field of cryo-electron microscopy (cryoEM) have enabled researchers to crack the structure of proteins that has been refractory to traditional crystallography techniques. High molecular weight kininogen is a good candidate for structural investigation by cryoEM. The goal of this review is to summarize the findings of kininogen structural studies.


Subject(s)
Kininogen, High-Molecular-Weight/genetics , Kininogen, High-Molecular-Weight/metabolism , Kininogen, High-Molecular-Weight/physiology , Animals , Bradykinin/metabolism , Cryoelectron Microscopy/methods , Humans , Kallikreins/blood , Kininogens/genetics , Kininogens/metabolism , Kininogens/physiology , Structure-Activity Relationship
8.
Proc Natl Acad Sci U S A ; 118(41)2021 10 12.
Article in English | MEDLINE | ID: covidwho-1462069

ABSTRACT

We describe a general method that allows structure determination of small proteins by single-particle cryo-electron microscopy (cryo-EM). The method is based on the availability of a target-binding nanobody, which is then rigidly attached to two scaffolds: 1) a Fab fragment of an antibody directed against the nanobody and 2) a nanobody-binding protein A fragment fused to maltose binding protein and Fab-binding domains. The overall ensemble of ∼120 kDa, called Legobody, does not perturb the nanobody-target interaction, is easily recognizable in EM images due to its unique shape, and facilitates particle alignment in cryo-EM image processing. The utility of the method is demonstrated for the KDEL receptor, a 23-kDa membrane protein, resulting in a map at 3.2-Šoverall resolution with density sufficient for de novo model building, and for the 22-kDa receptor-binding domain (RBD) of SARS-CoV-2 spike protein, resulting in a map at 3.6-Šresolution that allows analysis of the binding interface to the nanobody. The Legobody approach thus overcomes the current size limitations of cryo-EM analysis.


Subject(s)
Cryoelectron Microscopy/methods , SARS-CoV-2/metabolism , Single-Domain Antibodies/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Binding Sites/immunology , COVID-19/virology , HEK293 Cells , Humans , Models, Molecular , Protein Binding , Protein Domains , SARS-CoV-2/immunology , SARS-CoV-2/physiology , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/ultrastructure
9.
Mol Microbiol ; 117(3): 610-617, 2022 03.
Article in English | MEDLINE | ID: covidwho-1443320

ABSTRACT

Electron cryo-microscopy (cryo-EM) has lately emerged as a powerful method in structural biology and cell biology. While cryo-EM single-particle analysis (SPA) is now routinely delivering structures of purified proteins and protein complexes at near-atomic resolution, the use of electron cryo-tomography (cryo-ET), together with subtomogram averaging, is allowing visualization of macromolecular complexes in their native cellular environment, at unprecedented resolution. The unique ability of cryo-EM to provide information at many spatial resolution scales from ångströms to microns makes it an invaluable tool that bridges the classic "resolution-gap" between structural biology and cell biology domains. Like in many other fields of biology, in recent years, cryo-EM has revolutionized our understanding of pathogen biology, host-pathogen interaction and has made significant strides toward structure-based drug discovery. In a very recent example, during the ongoing coronavirus disease (COVID-19) pandemic, the structure of the stabilized severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein was deciphered by SPA. This led to the development of multiple vaccines. Alongside, cryo-ET provided key insights into the structure of the native virion, mechanism of its entry, replication, and budding; demonstrating the unrivaled power of cryo-EM in investigating pathogen biology, host-pathogen interaction, and drug discovery. In this review, we showcase a few examples of how different imaging modalities within cryo-EM have enabled the study of microbiology and host-pathogen interaction.


Subject(s)
COVID-19 , SARS-CoV-2 , Biology , Cryoelectron Microscopy/methods , Drug Discovery , Host-Pathogen Interactions , Humans
10.
Cell Rep ; 37(2): 109822, 2021 10 12.
Article in English | MEDLINE | ID: covidwho-1433046

ABSTRACT

Potent neutralizing monoclonal antibodies are one of the few agents currently available to treat COVID-19. SARS-CoV-2 variants of concern (VOCs) that carry multiple mutations in the viral spike protein can exhibit neutralization resistance, potentially affecting the effectiveness of some antibody-based therapeutics. Here, the generation of a diverse panel of 91 human, neutralizing monoclonal antibodies provides an in-depth structural and phenotypic definition of receptor binding domain (RBD) antigenic sites on the viral spike. These RBD antibodies ameliorate SARS-CoV-2 infection in mice and hamster models in a dose-dependent manner and in proportion to in vitro, neutralizing potency. Assessing the effect of mutations in the spike protein on antibody recognition and neutralization highlights both potent single antibodies and stereotypic classes of antibodies that are unaffected by currently circulating VOCs, such as B.1.351 and P.1. These neutralizing monoclonal antibodies and others that bind analogous epitopes represent potentially useful future anti-SARS-CoV-2 therapeutics.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , Antibodies, Neutralizing/immunology , SARS-CoV-2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Angiotensin-Converting Enzyme 2/ultrastructure , Animals , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/therapeutic use , Antibodies, Neutralizing/ultrastructure , Antibodies, Viral/immunology , COVID-19/immunology , Cricetinae , Cryoelectron Microscopy/methods , Epitopes/immunology , Female , Humans , Male , Mice , Mice, Inbred C57BL , Middle Aged , Neutralization Tests , Protein Binding/physiology , Receptors, Virus/metabolism , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
11.
Gene ; 808: 145963, 2022 Jan 15.
Article in English | MEDLINE | ID: covidwho-1415409

ABSTRACT

As of July 2021, the outbreak of coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, has led to more than 200 million infections and more than 4.2 million deaths globally. Complications of severe COVID-19 include acute kidney injury, liver dysfunction, cardiomyopathy, and coagulation dysfunction. Thus, there is an urgent need to identify proteins and genetic factors associated with COVID-19 susceptibility and outcome. We comprehensively reviewed recent findings of host-SARS-CoV-2 interactome analyses. To identify genetic variants associated with COVID-19, we focused on the findings from genome and transcriptome wide association studies (GWAS and TWAS) and bioinformatics analysis. We described established human proteins including ACE2, TMPRSS2, 40S ribosomal subunit, ApoA1, TOM70, HLA-A, and PALS1 interacting with SARS-CoV-2 based on cryo-electron microscopy results. Furthermore, we described approximately 1000 human proteins showing evidence of interaction with SARS-CoV-2 and highlighted host cellular processes such as innate immune pathways affected by infection. We summarized the evidence on more than 20 identified candidate genes in COVID-19 severity. Predicted deleterious and disruptive genetic variants with possible effects on COVID-19 infectivity have been also summarized. These findings provide novel insights into SARS-CoV-2 biology and infection as well as potential strategies for development of novel COVID therapeutic targets and drug repurposing.


Subject(s)
COVID-19/metabolism , Host Microbial Interactions/genetics , SARS-CoV-2/metabolism , COVID-19/physiopathology , Computational Biology/methods , Cryoelectron Microscopy/methods , Crystallography, X-Ray/methods , Genome-Wide Association Study , Host Microbial Interactions/physiology , Host-Pathogen Interactions/genetics , Humans , Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity
12.
Nat Struct Mol Biol ; 28(9): 747-754, 2021 09.
Article in English | MEDLINE | ID: covidwho-1370728

ABSTRACT

Drug discovery campaigns against COVID-19 are beginning to target the SARS-CoV-2 RNA genome. The highly conserved frameshift stimulation element (FSE), required for balanced expression of viral proteins, is a particularly attractive SARS-CoV-2 RNA target. Here we present a 6.9 Å resolution cryo-EM structure of the FSE (88 nucleotides, ~28 kDa), validated through an RNA nanostructure tagging method. The tertiary structure presents a topologically complex fold in which the 5' end is threaded through a ring formed inside a three-stem pseudoknot. Guided by this structure, we develop antisense oligonucleotides that impair FSE function in frameshifting assays and knock down SARS-CoV-2 virus replication in A549-ACE2 cells at 100 nM concentration.


Subject(s)
COVID-19/prevention & control , Cryoelectron Microscopy/methods , Frameshift Mutation/genetics , Oligonucleotides, Antisense/genetics , RNA, Viral/genetics , Response Elements/genetics , SARS-CoV-2/genetics , A549 Cells , Animals , Base Sequence , COVID-19/virology , Cell Line, Tumor , Chlorocebus aethiops , Genome, Viral/genetics , Humans , Models, Molecular , Nucleic Acid Conformation , Oligonucleotides, Antisense/pharmacology , RNA, Viral/chemistry , RNA, Viral/ultrastructure , SARS-CoV-2/physiology , SARS-CoV-2/ultrastructure , Vero Cells , Virus Replication/drug effects , Virus Replication/genetics
13.
J Vis Exp ; (173)2021 07 29.
Article in English | MEDLINE | ID: covidwho-1359306

ABSTRACT

In the past several years, technological and methodological advancements in single-particle cryo-electron microscopy (cryo-EM) have paved a new avenue for the high-resolution structure determination of biological macromolecules. Despite the remarkable advances in cryo-EM, there is still scope for improvement in various aspects of the single-particle analysis workflow. Single-particle analysis demands a suitable software package for high-throughput automatic data acquisition. Several automatic data acquisition software packages were developed for automatic imaging for single-particle cryo-EM in the last eight years. This paper presents an application of a fully automated image acquisition pipeline for vitrified biomolecules under low-dose conditions. It demonstrates a software package, which can collect cryo-EM data fully, automatically, and precisely. Additionally, various microscopic parameters are easily controlled by this software package. This protocol demonstrates the potential of this software package in automated imaging of the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) spike protein with a 200 keV cryo-electron microscope equipped with a direct electron detector (DED). Around 3,000 cryo-EM movie images were acquired in a single session (48 h) of data collection, yielding an atomic-resolution structure of the spike protein of SARS-CoV-2. Furthermore, this structural study indicates that the spike protein adopts two major conformations, 1-RBD (receptor-binding domain) up open and all RBD down closed conformations.


Subject(s)
COVID-19 , Cryoelectron Microscopy , Image Processing, Computer-Assisted , Software , Cryoelectron Microscopy/methods , Humans , SARS-CoV-2 , Spike Glycoprotein, Coronavirus
14.
Adv Mater ; 33(37): e2103221, 2021 Sep.
Article in English | MEDLINE | ID: covidwho-1321675

ABSTRACT

Liquid-phase electron microscopy (LP-EM) is an exciting new area in the materials imaging field, providing unprecedented views of molecular processes. Time-resolved insights from LP-EM studies are a strong complement to the remarkable results achievable with other high-resolution techniques. Here, the opportunities to expand LP-EM technology beyond 2D temporal assessments and into the 3D regime are described. The results show new structures and dynamic insights of human viruses contained in minute volumes of liquid while acquired in a rapid timeframe. To develop this strategy, adeno-associated virus (AAV) is used as a model system. AAV is a well-known gene therapy vehicle with current applications involving drug delivery and vaccine development for COVID-19. Improving the understanding of the physical properties of biological entities in a liquid state, as maintained in the human body, has broad societal implications for human health and disease.


Subject(s)
Cryoelectron Microscopy/methods , Dependovirus , Particle Size , COVID-19 , COVID-19 Vaccines , Drug Delivery Systems , Equipment Design , Genetic Therapy , HEK293 Cells/virology , Humans , Hydrogen-Ion Concentration , Immunoglobulin G/chemistry , Materials Testing , SARS-CoV-2
15.
Viruses ; 13(6)2021 06 11.
Article in English | MEDLINE | ID: covidwho-1270123

ABSTRACT

The SARS-CoV-2 virus has now become one of the greatest causes of infectious death and morbidity since the 1918 flu pandemic. Substantial and unprecedented progress has been made in the elucidation of the viral infection process in a short time; however, our understanding of the structure-function dynamics of the spike protein during the membrane fusion process and viral uptake remains incomplete. Employing computational approaches, we use full-length structural models of the SARS-CoV-2 spike protein integrating Cryo-EM images and biophysical properties, which fill the gaps in our understanding. We propose a membrane fusion model incorporating structural transitions associated with the proteolytic processing of the spike protein, which initiates and regulates a series of events to facilitate membrane fusion and viral genome uptake. The membrane fusion mechanism highlights the notable role of the S1 subunit and eventual mature spike protein uptake through the host membrane. Our comprehensive view accounts for distinct neutralizing antibody binding effects targeting the spike protein and the enhanced infectivity of the SARS-CoV-2 variant.


Subject(s)
Computer Simulation , Membrane Fusion , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization , Cryoelectron Microscopy/methods , Humans , Proteolysis , SARS-CoV-2/physiology
16.
Nat Commun ; 12(1): 3399, 2021 06 07.
Article in English | MEDLINE | ID: covidwho-1260942

ABSTRACT

Structures of macromolecular assemblies derived from cryo-EM maps often contain errors that become more abundant with decreasing resolution. Despite efforts in the cryo-EM community to develop metrics for map and atomistic model validation, thus far, no specific scoring metrics have been applied systematically to assess the interface between the assembly subunits. Here, we comprehensively assessed protein-protein interfaces in macromolecular assemblies derived by cryo-EM. To this end, we developed Protein Interface-score (PI-score), a density-independent machine learning-based metric, trained using the features of protein-protein interfaces in crystal structures. We evaluated 5873 interfaces in 1053 PDB-deposited cryo-EM models (including SARS-CoV-2 complexes), as well as the models submitted to CASP13 cryo-EM targets and the EM model challenge. We further inspected the interfaces associated with low-scores and found that some of those, especially in intermediate-to-low resolution (worse than 4 Å) structures, were not captured by density-based assessment scores. A combined score incorporating PI-score and fit-to-density score showed discriminatory power, allowing our method to provide a powerful complementary assessment tool for the ever-increasing number of complexes solved by cryo-EM.


Subject(s)
Cryoelectron Microscopy/methods , Macromolecular Substances/chemistry , Protein Interaction Domains and Motifs , Protein Interaction Mapping/methods , Protein Interaction Maps , Proteins/chemistry , Humans , Machine Learning , Macromolecular Substances/metabolism , Macromolecular Substances/ultrastructure , Models, Molecular , Neural Networks, Computer , Protein Conformation , Protein Multimerization , Proteins/metabolism , Proteins/ultrastructure , Support Vector Machine , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Viral Nonstructural Proteins/ultrastructure
17.
Proc Natl Acad Sci U S A ; 118(19)2021 05 11.
Article in English | MEDLINE | ID: covidwho-1254144

ABSTRACT

Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA-protein cross-linking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3' segment of the product RNA generated by backtracking extrudes through the RdRp nucleoside triphosphate (NTP) entry tunnel, that a mismatched nucleotide at the product RNA 3' end frays and enters the NTP entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals.


Subject(s)
COVID-19/virology , SARS-CoV-2/physiology , Virus Replication/genetics , Adenosine Monophosphate/pharmacology , Antiviral Agents/pharmacology , COVID-19/genetics , COVID-19/metabolism , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Cryoelectron Microscopy/methods , DNA Helicases/metabolism , Genome, Viral , Humans , Molecular Dynamics Simulation , RNA Helicases/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/metabolism , RNA-Dependent RNA Polymerase/physiology , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Viral Nonstructural Proteins/genetics
19.
Nat Methods ; 18(5): 439, 2021 05.
Article in English | MEDLINE | ID: covidwho-1242026
20.
Nat Methods ; 18(5): 442-443, 2021 05.
Article in English | MEDLINE | ID: covidwho-1242025
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