We report the crystal structure of the SARS-CoV-2 putative primase composed of the nsp7 and nsp8 proteins. We observed a dimer of dimers (2:2 nsp7-nsp8) in the crystallographic asymmetric unit. The structure revealed a fold with a helical core of the heterotetramer formed by both nsp7 and nsp8 that is flanked with two symmetry-related nsp8 β-sheet subdomains. It was also revealed that two hydrophobic interfaces one of approx. 1340 Å2 connects the nsp7 to nsp8 and a second one of approx. 950 Å2 connects the dimers and form the observed heterotetramer. Interestingly, analysis of the surface electrostatic potential revealed a putative RNA binding site that is formed only within the heterotetramer.

Institute of Organic Chemistry and Biochemistry AS CR v v i Flemingovo nam 2 166 10 Prague 6 Czech Republic

Zobrazit více v PubMed

Adams P.D., Afonine P.V., Bunkoczi G., Chen V.B., Davis I.W., Echols N., Headd J.J., Hung L.W., Kapral G.J., Grosse-Kunstleve R.W., McCoy A.J., Moriarty N.W., Oeffner R., Read R.J., Richardson D.C., Richardson J.S., Terwilliger T.C., Zwart P.H. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. Section D, Biol. Crystallogr. 2010;66:213–221. PubMed PMC

Afonine P.V., Grosse-Kunstleve R.W., Echols N., Headd J.J., Moriarty N.W., Mustyakimov M., Terwilliger T.C., Urzhumtsev A., Zwart P.H., Adams P.D. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. Section D, Biol. Crystallogr. 2012;68:352–367. PubMed PMC

Chu D.K., Leung C.Y., Gilbert M., Joyner P.H., Ng E.M., Tse T.M., Guan Y., Peiris J.S., Poon L.L. Avian coronavirus in wild aquatic birds. J. Virol. 2011;85:12815–12820. PubMed PMC

Coronaviridae Study Group of the International Committee on Taxonomy of, V., 2020. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol 5, 536-544. PubMed PMC

Dubankova A., Boura E. Structure of the yellow fever NS5 protein reveals conserved drug targets shared among flaviviruses. Antiviral Res. 2019;169 PubMed

Dubankova A., Humpolickova J., Klima M., Boura E. Negative charge and membrane-tethered viral 3B cooperate to recruit viral RNA dependent RNA polymerase 3D (pol) Sci. Rep. 2017;7:17309. PubMed PMC

Emsley P., Lohkamp B., Scott W.G., Cowtan K. Features and development of Coot. Acta Crystallogr. Section D, Biol. Crystallogr. 2010;66:486–501. PubMed PMC

Gao, Y., Yan, L., Huang, Y., Liu, F., Zhao, Y., Cao, L., Wang, T., Sun, Q., Ming, Z., Zhang, L., Ge, J., Zheng, L., Zhang, Y., Wang, H., Zhu, Y., Zhu, C., Hu, T., Hua, T., Zhang, B., Yang, X., Li, J., Yang, H., Liu, Z., Xu, W., Guddat, L.W., Wang, Q., Lou, Z., Rao, Z., 2020. Structure of the RNA-dependent RNA polymerase from COVID-19 virus. Science. PubMed PMC

Gouet P., Courcelle E., Stuart D.I., Metoz F. ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics. 1999;15:305–308. PubMed

Gralinski, L.E., Menachery, V.D., 2020. Return of the Coronavirus: 2019-nCoV. Viruses 12. PubMed PMC

Hercik K., Kozak J., Sala M., Dejmek M., Hrebabecky H., Zbornikova E., Smola M., Ruzek D., Nencka R., Boura E. Adenosine triphosphate analogs can efficiently inhibit the Zika virus RNA-dependent RNA polymerase. Antiviral Res. 2017;137:131–133. PubMed

Hillen H.S., Kokic G., Farnung L., Dienemann C., Tegunov D., Cramer P. Structure of replicating SARS-CoV-2 polymerase. Nature. 2020 PubMed

Huang C., Wang Y., Li X., Ren L., Zhao J., Hu Y., Zhang L., Fan G., Xu J., Gu X., Cheng Z., Yu T., Xia J., Wei Y., Wu W., Xie X., Yin W., Li H., Liu M., Xiao Y., Gao H., Guo L., Xie J., Wang G., Jiang R., Gao Z., Jin Q., Wang J., Cao B. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020;395:497–506. PubMed PMC

Imbert I., Guillemot J.C., Bourhis J.M., Bussetta C., Coutard B., Egloff M.P., Ferron F., Gorbalenya A.E., Canard B. A second, non-canonical RNA-dependent RNA polymerase in SARS coronavirus. EMBO J. 2006;25:4933–4942. PubMed PMC

Kabsch W. Xds. Acta Crystallogr. Section D, Biol. Crystallogr. 2010;66:125–132. PubMed PMC

Kirchdoerfer R.N., Ward A.B. Structure of the SARS-CoV nsp12 polymerase bound to nsp7 and nsp8 co-factors. Nat. Commun. 2019;10:2342. PubMed PMC

Ksiazek, T.G., Erdman, D., Goldsmith, C.S., Zaki, S.R., Peret, T., Emery, S., Tong, S., Urbani, C., Comer, J.A., Lim, W., Rollin, P.E., Dowell, S.F., Ling, A.E., Humphrey, C.D., Shieh, W.J., Guarner, J., Paddock, C.D., Rota, P., Fields, B., DeRisi, J., Yang, J.Y., Cox, N., Hughes, J.M., LeDuc, J.W., Bellini, W.J., Anderson, L.J., Group, S.W., 2003. A novel coronavirus associated with severe acute respiratory syndrome The New England journal of medicine 348 1953 1966. PubMed

McCoy A.J., Grosse-Kunstleve R.W., Adams P.D., Winn M.D., Storoni L.C., Read R.J. Phaser crystallographic software. J. Appl. Crystallogr. 2007;40:658–674. PubMed PMC

Perlman S., Netland J. Coronaviruses post-SARS: update on replication and pathogenesis. Nat Rev Microbiol. 2009;7:439–450. PubMed PMC

Rota P.A., Oberste M.S., Monroe S.S., Nix W.A., Campagnoli R., Icenogle J.P., Penaranda S., Bankamp B., Maher K., Chen M.H., Tong S., Tamin A., Lowe L., Frace M., DeRisi J.L., Chen Q., Wang D., Erdman D.D., Peret T.C., Burns C., Ksiazek T.G., Rollin P.E., Sanchez A., Liffick S., Holloway B., Limor J., McCaustland K., Olsen-Rasmussen M., Fouchier R., Gunther S., Osterhaus A.D., Drosten C., Pallansch M.A., Anderson L.J., Bellini W.J. Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science. 2003;300:1394–1399. PubMed

Sebera J., Dubankova A., Sychrovsky V., Ruzek D., Boura E., Nencka R. The structural model of Zika virus RNA-dependent RNA polymerase in complex with RNA for rational design of novel nucleotide inhibitors. Sci. Rep. 2018;8:11132. PubMed PMC

Snijder E.J., Decroly E., Ziebuhr J. The nonstructural proteins directing coronavirus RNA synthesis and processing. Adv. Virus Res. 2016;96:59–126. PubMed PMC

te Velthuis A.J., van den Worm S.H., Snijder E.J. The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension. Nucl. Acids Res. 2012;40:1737–1747. PubMed PMC

Thompson J.D., Higgins D.G., Gibson T.J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl. Acids Res. 1994;22:4673–4680. PubMed PMC

Torres C.A., Hora A.S., Tonietti P.O., Taniwaki S.A., Cecchinato M., Villarreal L.Y., Brandao P.E. Gammacoronavirus and deltacoronavirus in quail. Avian Dis. 2016;60:656–661. PubMed

Wang L., Byrum B., Zhang Y. Detection and genetic characterization of deltacoronavirus in pigs, Ohio, USA, 2014. Emerg. Infect. Dis. 2014;20:1227–1230. PubMed PMC

Woo P.C., Lau S.K., Lam C.S., Lau C.C., Tsang A.K., Lau J.H., Bai R., Teng J.L., Tsang C.C., Wang M., Zheng B.J., Chan K.H., Yuen K.Y. Discovery of seven novel Mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus. J. Virol. 2012;86:3995–4008. PubMed PMC

Wu A., Peng Y., Huang B., Ding X., Wang X., Niu P., Meng J., Zhu Z., Zhang Z., Wang J., Sheng J., Quan L., Xia Z., Tan W., Cheng G., Jiang T. Genome composition and divergence of the novel coronavirus (2019-nCoV) originating in China. Cell Host Microbe. 2020;27:325–328. PubMed PMC

Xiao Y., Ma Q., Restle T., Shang W., Svergun D.I., Ponnusamy R., Sczakiel G., Hilgenfeld R. Nonstructural proteins 7 and 8 of feline coronavirus form a 2:1 heterotrimer that exhibits primer-independent RNA polymerase activity. J. Virol. 2012;86:4444–4454. PubMed PMC

Zaki A.M., van Boheemen S., Bestebroer T.M., Osterhaus A.D., Fouchier R.A. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. New England J. Med. 2012;367:1814–1820. PubMed

Zhai Y., Sun F., Li X., Pang H., Xu X., Bartlam M., Rao Z. Insights into SARS-CoV transcription and replication from the structure of the nsp7-nsp8 hexadecamer. Nat. Struct. Mol. Biol. 2005;12:980–986. PubMed PMC

Zhu, N., Zhang, D., Wang, W., Li, X., Yang, B., Song, J., Zhao, X., Huang, B., Shi, W., Lu, R., Niu, P., Zhan, F., Ma, X., Wang, D., Xu, W., Wu, G., Gao, G.F., Tan, W., China Novel Coronavirus, I., Research, T., 2020. A Novel Coronavirus from Patients with Pneumonia in China, 2019. The New England journal of medicine 382, 727-733. PubMed PMC

Ziebuhr J. The coronavirus replicase. Curr. Top. Microbiol. Immunol. 2005;287:57–94. PubMed PMC

Zumla A., Hui D.S., Perlman S. Middle East respiratory syndrome. Lancet. 2015;386:995–1007. PubMed PMC

Zumla A., Chan J.F., Azhar E.I., Hui D.S., Yuen K.Y. Coronaviruses - drug discovery and therapeutic options. Nat. Rev. Drug Discov. 2016;15:327–347. PubMed PMC

The Present and Future of Virology in the Czech Republic-A New Phoenix Made of Ashes?

A Helquat-like Compound as a Potent Inhibitor of Flaviviral and Coronaviral Polymerases

Coronaviral RNA-methyltransferases: function, structure and inhibition

Substrate Specificity of SARS-CoV-2 Nsp10-Nsp16 Methyltransferase

Localization of SARS-CoV-2 Capping Enzymes Revealed by an Antibody against the nsp10 Subunit

Structural Analysis of the OC43 Coronavirus 2'-O-RNA Methyltransferase