Structure of the SARS-CoV NSP12 polymerase bound to NSP7 and NSP8 co-factors

Recent history is punctuated by the emergence of highly pathogenic coronaviruses such as SARS- and MERS-CoV into human circulation. Upon infecting host cells, coronaviruses assemble a multi-subunit RNA-synthesis complex of viral non-structural proteins (NSP) responsible for the replication and transcription of the viral genome. Here, we present the 3.1 Å resolution structure of the SARS-CoV NSP12 polymerase bound to its essential co-factors, NSP7 and NSP8, using single particle cryo-electron microscopy. NSP12 possesses an architecture common to all viral polymerases as well as a large N-terminal extension containing a kinase-like fold and is unexpectedly bound by two NSP8 co-factors. This structure illuminates the assembly of the coronavirus core RNA-synthesis machinery, provides key insights into NSP12 polymerase catalysis and fidelity and acts as a template for the design of novel antiviral therapeutics.

[1]  Christopher J. Williams,et al.  MolProbity: More and better reference data for improved all‐atom structure validation , 2018, Protein science : a publication of the Protein Society.

[2]  Anchi Cheng,et al.  Automated molecular microscopy: the new Leginon system. , 2005, Journal of structural biology.

[3]  Raymond C Stevens,et al.  Severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[4]  Update: severe acute respiratory syndrome--United States, May 21, 2003. , 2003, MMWR. Morbidity and mortality weekly report.

[5]  Dmitri I. Svergun,et al.  Nonstructural Proteins 7 and 8 of Feline Coronavirus Form a 2:1 Heterotrimer That Exhibits Primer-Independent RNA Polymerase Activity , 2012, Journal of Virology.

[6]  D. Agard,et al.  MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy , 2017, Nature Methods.

[7]  Nathaniel Echols,et al.  EMRinger: Side-chain-directed model and map validation for 3D Electron Cryomicroscopy , 2015, Nature Methods.

[8]  Kin Moy,et al.  Crystal Structure of Nonstructural Protein 10 from the Severe Acute Respiratory Syndrome Coronavirus Reveals a Novel Fold with Two Zinc-Binding Motifs , 2006, Journal of Virology.

[9]  M. Vignuzzi,et al.  Homology-Based Identification of a Mutation in the Coronavirus RNA-Dependent RNA Polymerase That Confers Resistance to Multiple Mutagens , 2016, Journal of Virology.

[10]  David J. Fleet,et al.  cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination , 2017, Nature Methods.

[11]  S. Gottlieb.  New York's war on tobacco produces record fall in smoking , 2004, BMJ : British Medical Journal.

[12]  Fabian Sievers,et al.  Clustal Omega for making accurate alignments of many protein sequences , 2018, Protein science : a publication of the Protein Society.

[13]  M. Denison,et al.  Characterization of the Expression, Intracellular Localization, and Replication Complex Association of the Putative Mouse Hepatitis Virus RNA-Dependent RNA Polymerase , 2003, Journal of Virology.

[14]  Liisa Holm,et al.  Dali server update , 2016, Nucleic Acids Res..

[15]  J. Epstein,et al.  Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor , 2013, Nature.

[16]  A. M. Leontovich,et al.  Discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the RNA polymerase-containing protein of all nidoviruses , 2015, Nucleic acids research.

[17]  S. M. McDonald.  RNA synthetic mechanisms employed by diverse families of RNA viruses , 2013, Wiley interdisciplinary reviews. RNA.

[18]  E. Decroly,et al.  One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities , 2014, Proceedings of the National Academy of Sciences.

[19]  M. Rossmann,et al.  RNA-dependent RNA polymerases from Flaviviridae. , 2009, Current opinion in structural biology.

[20]  Liming Yan,et al.  Structural basis and functional analysis of the SARS coronavirus nsp14–nsp10 complex , 2015, Proceedings of the National Academy of Sciences.

[21]  F. Rey,et al.  Structural Analysis of the Hepatitis C Virus RNA Polymerase in Complex with Ribonucleotides , 2002, Journal of Virology.

[22]  J. Bruenn.  A structural and primary sequence comparison of the viral RNA-dependent RNA polymerases. , 2003, Nucleic acids research.

[23]  M Radermacher,et al.  DoG Picker and TiltPicker: software tools to facilitate particle selection in single particle electron microscopy. , 2009, Journal of structural biology.

[24]  Yu Chen,et al.  Molecular mechanisms of coronavirus RNA capping and methylation , 2016, Virologica Sinica.

[25]  L. van der Hoek.  Human Coronaviruses: What Do They Cause? , 2005, Antiviral therapy.

[26]  J. Parry.  Breaches of safety regulations are probable cause of recent SARS outbreak, WHO says , 2004, BMJ : British Medical Journal.

[27]  B. Coutard,et al.  A second, non-canonical RNA-dependent RNA polymerase in SARS Coronavirus , 2006, The EMBO journal.

[28]  I Sauvaget,et al.  Identification of four conserved motifs among the RNA‐dependent polymerase encoding elements. , 1989, The EMBO journal.

[29]  Caroline C. Friedel,et al.  Analysis of Intraviral Protein-Protein Interactions of the SARS Coronavirus ORFeome , 2007, PloS one.

[30]  J. Choi,et al.  Biochemical characterization of a recombinant SARS coronavirus nsp12 RNA-dependent RNA polymerase capable of copying viral RNA templates , 2012, Archives of Virology.

[31]  Xi Lin,et al.  Regulation of polarized extension and planar cell polarity in the cochlea by the vertebrate PCP pathway , 2005, Nature Genetics.

[32]  Zihe Rao,et al.  Insights into SARS-CoV transcription and replication from the structure of the nsp7–nsp8 hexadecamer , 2005, Nature Structural &Molecular Biology.

[33]  D. Falzarano,et al.  SARS and MERS: recent insights into emerging coronaviruses , 2016, Nature Reviews Microbiology.

[34]  Matthias J. Brunner,et al.  Atomic accuracy models from 4.5 Å cryo-electron microscopy data with density-guided iterative local refinement , 2015, Nature Methods.

[35]  Dahai Luo,et al.  A Crystal Structure of the Dengue Virus NS5 Protein Reveals a Novel Inter-domain Interface Essential for Protein Flexibility and Virus Replication , 2015, PLoS pathogens.

[36]  Eric J. Snijder,et al.  The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension , 2011, Nucleic acids research.

[37]  Xiaotao Lu,et al.  Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease , 2018, mBio.

[38]  C. Cameron,et al.  The Palm Subdomain-based Active Site is Internally Permuted in Viral RNA-dependent RNA Polymerases of an Ancient Lineage , 2002, Journal of Molecular Biology.

[39]  Andrew B. Ward,et al.  EMHP: an accurate automated hole masking algorithm for single-particle cryo-EM image processing , 2017, bioRxiv.

[40]  Kai Zhang,et al.  Gctf: Real-time CTF determination and correction , 2015, bioRxiv.

[41]  E. Decroly,et al.  Insights into RNA synthesis, capping, and proofreading mechanisms of SARS-coronavirus , 2014, Virus Research.

[42]  D. Liu,et al.  Coronavirus infectious bronchitis virus non-structural proteins 8 and 12 form stable complex independent of the non-translated regions of viral RNA and other viral proteins , 2017, Virology.

[43]  Randy J. Read,et al.  Acta Crystallographica Section D Biological , 2003 .

[44]  P. Dragovich,et al.  The crystal structure of the RNA-dependent RNA polymerase from human rhinovirus: a dual function target for common cold antiviral therapy. , 2004, Structure.

[45]  J. Joseph,et al.  Atlas of coronavirus replicase structure , 2013, Virus Research.

[46]  Itay Mayrose,et al.  ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules , 2016, Nucleic Acids Res..

[47]  K. Ng,et al.  Binding of 2'-amino-2'-deoxycytidine-5'-triphosphate to norovirus polymerase induces rearrangement of the active site. , 2009, Journal of molecular biology.

[48]  J. Ziebuhr.  The Coronavirus Replicase , 2005, Current topics in microbiology and immunology.

[49]  Erik Lindahl,et al.  New tools for automated high-resolution cryo-EM structure determination in RELION-3 , 2018, eLife.

[50]  Gabriel C Lander,et al.  Achieving better than 3 Å resolution by single particle cryo-EM at 200 keV , 2017, Nature Methods.

[51]  Gérard Bricogne,et al.  Crystal Structure and Functional Analysis of the SARS-Coronavirus RNA Cap 2′-O-Methyltransferase nsp10/nsp16 Complex , 2011, PLoS pathogens.

[52]  Rong Zhao,et al.  Crystal structure of Middle East respiratory syndrome coronavirus helicase , 2017, PLoS pathogens.

[53]  P. Emsley,et al.  Features and development of Coot , 2010, Acta crystallographica. Section D, Biological crystallography.

[54]  M. Denison,et al.  Coronaviruses as DNA Wannabes: A New Model for the Regulation of RNA Virus Replication Fidelity , 2013, PLoS pathogens.

[55]  O. Peersen.  Picornaviral polymerase structure, function, and fidelity modulation. , 2017, Virus research.

[56]  O. Peersen,et al.  Structural basis for active site closure by the poliovirus RNA-dependent RNA polymerase , 2010, Proceedings of the National Academy of Sciences.

[57]  David I Stuart,et al.  The nsp9 Replicase Protein of SARS-Coronavirus, Structure and Functional Insights , 2004, Structure.