Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form

SARS-CoV-2 ORF7b: is a bat virus protein homologue a major cause of COVID-19 symptoms?

Abstract ORF7b is an accessory protein of SARS-CoV-2, the virus behind the COVID-19 pandemic. Using cell-free synthesized ORF7b, we experimentally show that ORF7b assembles into stable multimers. The ORF7b sequence shows a transmembrane segment, which multimerizes through a leucine zipper. We hypothesize that ORF7b has the potential to interfere with important cellular processes that involve leucine-zipper formation, and present two particularly striking examples. First, leucine zippers are central in heart rhythm regulation through multimerization of phospholamban in cardiomyocytes. Second, epithelial cell-cell adhesion relies on E-cadherins, which dimerize using a transmembrane leucine zipper. Most common symptoms of SARS-CoV-2 infection, including heart arrythmias, odor loss, impaired oxygen uptake and intestinal problems, up to multiorgan failure, can be rationalized by a possible interference of ORF7b with the functions of these proteins. We ask whether this is pure coincidence, or whether our observations point to disruption by ORF7b of vital processes in COVID-19

Molecular elucidation of drug-induced abnormal assemblies of the hepatitis B virus capsid protein by solid-state NMR

Hepatitis B virus (HBV) capsid assembly modulators (CAMs) represent a recent class of anti-HBV antivirals. CAMs disturb proper nucleocapsid assembly, by inducing formation of either aberrant assemblies (CAM-A) or of apparently normal but genome-less empty capsids (CAM-E). Classical structural approaches have revealed the CAM binding sites on the capsid protein (Cp), but conformational information on the CAM-induced off-path aberrant assemblies is lacking. Here we show that solid-state NMR can provide such information, including for wild-type full-length Cp183, and we find that in these assemblies, the asymmetric unit comprises a single Cp molecule rather than the four quasi-equivalent conformers typical for the icosahedral T = 4 symmetry of the normal HBV capsids. Furthermore, while in contrast to truncated Cp149, full-length Cp183 assemblies appear, on the mesoscopic level, unaffected by CAM-A, NMR reveals that on the molecular level, Cp183 assemblies are equally aberrant. Finally, we use a eukaryotic cell-free system to reveal how CAMs modulate capsid-RNA interactions and capsid phosphorylation. Our results establish a structural view on assembly modulation of the HBV capsid, and they provide a rationale for recently observed differences between in-cell versus in vitro capsid assembly modulation.ISSN:2041-172

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.This work was supported by Goethe University (Corona funds), the DFG-funded CRC: “Molecular Principles of RNA-Based Regulation,” DFG infrastructure funds (project numbers: 277478796, 277479031, 392682309, 452632086, 70653611), the state of Hesse (BMRZ), the Fondazione CR Firenze (CERM), and the IWB-EFRE-program 20007375. This project has received funding from the European Union’s Horizon 2020 research and innovation program under Grant Agreement No. 871037. AS is supported by DFG Grant SCHL 2062/2-1 and by the JQYA at Goethe through project number 2019/AS01. Work in the lab of KV was supported by a CoRE grant from the University of New Hampshire. The FLI is a member of the Leibniz Association (WGL) and financially supported by the Federal Government of Germany and the State of Thuringia. Work in the lab of RM was supported by NIH (2R01EY021514) and NSF (DMR-2002837). BN-B was supported by theNSF GRFP.MCwas supported byNIH (R25 GM055246 MBRS IMSD), and MS-P was supported by the HHMI Gilliam Fellowship. Work in the labs of KJ and KT was supported by Latvian Council of Science Grant No. VPP-COVID 2020/1-0014. Work in the UPAT’s lab was supported by the INSPIRED (MIS 5002550) project, which is implemented under the Action “Reinforcement of the Research and Innovation Infrastructure,” funded by the Operational Program “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and cofinanced by Greece and the EU (European Regional Development Fund) and the FP7 REGPOT CT-2011- 285950–“SEE-DRUG” project (purchase of UPAT’s 700MHz NMR equipment). Work in the CM-G lab was supported by the Helmholtz society. Work in the lab of ABö was supported by the CNRS, the French National Research Agency (ANR, NMRSCoV2- ORF8), the Fondation de la Recherche Médicale (FRM, NMR-SCoV2-ORF8), and the IR-RMN-THC Fr3050 CNRS. Work in the lab of BM was supported by the Swiss National Science Foundation (Grant number 200020_188711), the Günthard Stiftung für Physikalische Chemie, and the ETH Zurich. Work in the labs of ABö and BM was supported by a common grant from SNF (grant 31CA30_196256). This work was supported by the ETHZurich, the grant ETH40 18 1, and the grant Krebsliga KFS 4903 08 2019. Work in the lab of the IBS Grenoble was supported by the Agence Nationale de Recherche (France) RA-COVID SARS2NUCLEOPROTEIN and European Research Council Advanced Grant DynamicAssemblies. Work in the CA lab was supported by Patto per il Sud della Regione Siciliana–CheMISt grant (CUP G77B17000110001). Part of this work used the platforms of the Grenoble Instruct-ERIC center (ISBG; UMS 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-05-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE- 0003). Work at the UW-Madison was supported by grant numbers NSF MCB2031269 and NIH/NIAID AI123498. MM is a Ramón y Cajal Fellow of the Spanish AEI-Ministry of Science and Innovation (RYC2019-026574-I), and a “La Caixa” Foundation (ID 100010434) Junior Leader Fellow (LCR/BQ/PR19/11700003). Funded by project COV20/00764 fromthe Carlos III Institute of Health and the SpanishMinistry of Science and Innovation to MMand DVL. VDJ was supported by the Boehringer Ingelheim Fonds. Part of this work used the resources of the Italian Center of Instruct-ERIC at the CERM/ CIRMMP infrastructure, supported by the Italian Ministry for University and Research (FOE funding). CF was supported by the Stiftung Polytechnische Gesellschaft. Work in the lab of JH was supported by NSF (RAPID 2030601) and NIH (R01GM123249).Peer reviewe