vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/VJGB-22-15vavilov-3287Research ArticleМОЛЕКУЛЯРНАЯ И КЛЕТОЧНАЯ БИОЛОГИЯMOLECULAR AND CELL BIOLOGYРепликационно-транскрипционный комплекс коронавирусов: функции индивидуальных вирусных неструктурных субъединиц, свойства и архитектура их комплексовReplication-transcription complex of coronaviruses: functions of individual viral non-structural subunits, properties and architecture of their complexesМищенкоЕ. Л.MishchenkoE. L.

Новосибирск

Novosibirsk

elmish@bionet.nsc.ruИванисенкоВ. А.IvanisenkoV. A.

Новосибирск

Novosibirsk

Федеральный исследовательский центр Институт цитологии и генетики Сибирского отделения Российской академии наукРоссияInstitute of Cytology and Genetics of the Siberian Branch of the Russian Academy of SciencesRussian Federation202204042022262121127Copyright © Мищенко Е.Л., Иванисенко В.А., 20222022Мищенко Е.Л., Иванисенко В.А.Mishchenko E.L., Ivanisenko V.A.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/3287

Коронавирусы относятся к семейству Coronaviridae подсемейства Orthocoronavirinae и представляют собой оболочечные (+) РНК-вирусы с необычно длинным геномом. В настоящее время часто идентифицируются, вызывая продолжающуюся пандемию во всем мире, коронавирусы Severe Acute respiratory syndrome CoV (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV) и новый коронавирус (2019-nCoV, SARS-CoV-2). Для сдерживания быстро распространяющейся, острой и часто тяжело протекающей инфекции широко применяют прошедшие клинические испытания вакцины, однако эффективных лекарств по-прежнему нет. Геномы SARS-CoV-2 и SARS-CoV идентичны на ~80 %, а SARS-CoV-2 и MERS-CoV – на ~50 %. Это свидетельствует об общих механизмах патогенеза коронавирусов и одних и тех же потенциальных терапевтических мишенях. Ферменты и эффекторные белки, входящие в состав репликационно-транскрипционного комплек-са (РТК) коронавирусов, кодируются весьма крупным геном репликазы и представляют собой перспективные мишени действия потенциальных эффективных лекарств. Эти мишени включают папаин- и 3С-подобные цистеиновые протеиназы, осуществляющие процессинг двух больших вирусных полипротеинов, РНК-зависимую РНК-полимеразу, РНК-хеликазу, ферменты, модифицирующие вирусный геном, ферменты, обладающие 3’–5’-экзорибонуклеазной и уридилат-специфичной эндонуклеазной активностью, а также важные эффекторные белки. В настоящее время изучение сложных молекулярных механизмов сборки и функционирования РТК находится на пике изучения. Обзор посвящен актуальным и современным исследованиям свойств индивидуальных неструктурных субъединиц РТК и их комплексов и включает изучение структур индивидуальных субъединиц РТК коронавирусов, доменной организации субъединиц и их функций, белок-белковых взаимодействий, свойств и архитектуры комплексов субъединиц, влияния мутаций, а также выявления мутаций, влияющих на жизнеспособность вируса в клеточной культуре.

Coronaviruses (CoVs) belong to the subfamily Orthocoronavirinae of the family Coronaviridae. CoVs are enveloped (+) RNA viruses with unusually long genomes. Severe acute respiratory syndrome CoV (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and the novel coronavirus (2019-nCoV, SARS-CoV-2) have been identif ied as causing global pandemics. Clinically tested vaccines are widely used to control rapidly spreading, acute, and often severe infections; however, effective drugs are still not available. The genomes of SARS-CoV-2 and SARS-CoV are approximately 80 % identical, while the genomes of SARS-CoV-2 and MERS-CoV are approximately 50 % identical. This indicates that there may be common mechanisms of coronavirus pathogenesis and, therefore, potential therapeutic targets for each virus may be the same. The enzymes and effector proteins that make up the replicationtranscription complex (RTC) of coronaviruses are encoded by a large replicase gene. These enzymes and effector proteins represent promising targets for potential therapeutic drugs. The enzyme targets include papain- and 3C-like cysteine proteinases that process two large viral polyproteins, RNA-dependent RNA polymerase, RNA helicase, viral genome-modifying enzymes, and enzymes with 3’–5’ exoribonuclease or uridylate-specif ic endonuclease activity. Currently, there are many studies investigating the complex molecular mechanisms involved in the assembly and function of the RTC. This review will encompass current, modern studies on the properties and complexes of individual non-structural subunits of the RTC, the structures of individual coronavirus RTC subunits, domain organization and functions of subunits, protein-protein interactions, properties and architectures of subunit complexes, the effect of mutations, and the identif ication of mutations affecting the viability of the virus in cell culture.

неструктурные белки коронавирусов (CoVs)субъединицы репликазы CoVsрепликационнотранскрипционный комплекс CoVsархитектура комплексов неструктурных белков CoVsnon-structural proteins CoVssubunits of replicase CoVsreplication-transcription complex of CoVsarchitecture of non-structural protein complexes CoVs.The work was carried out with the support of budget project No. FWNR-2022-0020.ReferencesAdedeji A.O., Lazarus H. Biochemical characterization of Middle East respiratory syndrome coronavirus helicase. mSphere. 2016;1(5): e00235-16. DOI 10.1128/mSphere.00235-16.Adedeji A.O., Lazarus H. Biochemical characterization of Middle East respiratory syndrome coronavirus helicase. mSphere. 2016;1(5): e00235-16. DOI 10.1128/mSphere.00235-16.Adedeji A.O., Marchand B., Velthuis A.J.W., Snijder E.J., Weiss S., Eoff R.L., Singh K., Sarafianos S.G. Mechanism of nucleic acid unwinding by SARS-CoV helicase. PLoS One. 2012;7(5):e36521. DOI 10.1371/journal.pone.0036521.Adedeji A.O., Marchand B., Velthuis A.J.W., Snijder E.J., Weiss S., Eoff R.L., Singh K., Sarafianos S.G. Mechanism of nucleic acid unwinding by SARS-CoV helicase. PLoS One. 2012;7(5):e36521. DOI 10.1371/journal.pone.0036521.Angelini M.M., Akhlaghpour M., Neuman B.W., Buchmeier M.J. Severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles. mBio. 2013; 4(4):e00524-13. DOI 10.1128/mBio.00524-13.Angelini M.M., Akhlaghpour M., Neuman B.W., Buchmeier M.J. Severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles. mBio. 2013; 4(4):e00524-13. DOI 10.1128/mBio.00524-13.Bhardwaj K., Sun J., Holzenburg A., Guarino L.A., Kao C.C. RNA recognition and cleavage by the SARS coronavirus endoribonuclease. J. Mol. Biol. 2006;361(2):243-256. DOI 10.1016/j.jmb.2006. 06.021.Bhardwaj K., Sun J., Holzenburg A., Guarino L.A., Kao C.C. RNA recognition and cleavage by the SARS coronavirus endoribonuclease. J. Mol. Biol. 2006;361(2):243-256. DOI 10.1016/j.jmb.2006. 06.021.Bouvet M., Debarnot C., Imbert I., Selisko B., Snijder E.J., Canard B., Decroly E. In vitro reconstitution of SARS-coronavirus mRNA cap methylation. PLoS Pathog. 2010;6(4):e1000863. DOI 10.1371/journal.ppat.1000863.Bouvet M., Debarnot C., Imbert I., Selisko B., Snijder E.J., Canard B., Decroly E. In vitro reconstitution of SARS-coronavirus mRNA cap methylation. PLoS Pathog. 2010;6(4):e1000863. DOI 10.1371/journal.ppat.1000863.Bouvet M., Imbert I., Subissi L., Gluais L., Canard B., Decroly E. RNA 3′-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/nsp14 exoribonuclease complex. Proc. Natl. Acad. Sci. USA. 2012;109(24):9372-9377. DOI 10.1073/pnas.1201130109.Bouvet M., Imbert I., Subissi L., Gluais L., Canard B., Decroly E. RNA 3′-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/nsp14 exoribonuclease complex. Proc. Natl. Acad. Sci. USA. 2012;109(24):9372-9377. DOI 10.1073/pnas.1201130109.Bouvet M., Lugari A., Posthuma C.C., Zevenhoven J.C., Bernard S., Betzi S., Imbert I., Canard B., Guillemot J.-C., Lécine P., Pfefferle S., Drosten C., Snijder E.J., Decroly E., Morelli X. Coronavirus Nsp10, a critical co-factor for activation of multiple replicative enzymes. J. Biol. Chem. 2014;289(37):25783-25796. DOI 10.1074/jbc.M114.577353.Bouvet M., Lugari A., Posthuma C.C., Zevenhoven J.C., Bernard S., Betzi S., Imbert I., Canard B., Guillemot J.-C., Lécine P., Pfefferle S., Drosten C., Snijder E.J., Decroly E., Morelli X. Coronavirus Nsp10, a critical co-factor for activation of multiple replicative enzymes. J. Biol. Chem. 2014;289(37):25783-25796. DOI 10.1074/jbc.M114.577353.Brunn A., Teepe C., Simpson J.C., Pepperkok R., Friedel C.C., Zimmer R., Roberts R., Baric R., Haas J. Analysis of intraviral proteinprotein interactions of the SARS coronavirus ORFeome. PLoS One. 2007;2(5):e459. DOI 10.1371/journal.pone.0000459.Brunn A., Teepe C., Simpson J.C., Pepperkok R., Friedel C.C., Zimmer R., Roberts R., Baric R., Haas J. Analysis of intraviral proteinprotein interactions of the SARS coronavirus ORFeome. PLoS One. 2007;2(5):e459. DOI 10.1371/journal.pone.0000459.Chen J., Malone B., Llewellyn E., Grasso M., Shelton P.M.M., Olinares P.D.B., Maruthi K., Eng E.T., Vatandaslar H., Chait B.T., Kapoor T.M., Darst S.A., Campbell E.A. Structural basis for helicasepolymerase coupling in the SARS-CoV-2 replication-transcription complex. Cell. 2020;182(6):1560-1573.e13. DOI 10.1016/j.cell.2020.07.033.Chen J., Malone B., Llewellyn E., Grasso M., Shelton P.M.M., Olinares P.D.B., Maruthi K., Eng E.T., Vatandaslar H., Chait B.T., Kapoor T.M., Darst S.A., Campbell E.A. Structural basis for helicasepolymerase coupling in the SARS-CoV-2 replication-transcription complex. Cell. 2020;182(6):1560-1573.e13. DOI 10.1016/j.cell.2020.07.033.Chen Y., Cai H., Pan J., Xiang N., Tien P., Ahola T., Guo D. Functional screen reveals SARS coronavirus nonstructural protein nsp14 as a novel cap N7 methyltransferase. Proc. Natl. Acad. Sci. USA. 2009;106(9):3484-3489. DOI 10.1073/pnas.0808790106.Chen Y., Cai H., Pan J., Xiang N., Tien P., Ahola T., Guo D. Functional screen reveals SARS coronavirus nonstructural protein nsp14 as a novel cap N7 methyltransferase. Proc. Natl. Acad. Sci. USA. 2009;106(9):3484-3489. DOI 10.1073/pnas.0808790106.Cottam E.M., Maier H.J., Manifava M., Vaux L.C., Chandra-Schoenfelder P., Gerner W., Britton P., Ktistakis N.T., Wileman T. Coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate. Autophagy. 2011;7(11): 1335-1347. DOI 10.4161/auto.7.11.16642.Cottam E.M., Maier H.J., Manifava M., Vaux L.C., Chandra-Schoenfelder P., Gerner W., Britton P., Ktistakis N.T., Wileman T. Coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate. Autophagy. 2011;7(11): 1335-1347. DOI 10.4161/auto.7.11.16642.Daczkowski C.M., Dzimianski J.V., Clasman J.R., Goodwin O., Mesecar A.D., Pegan S.D. Structural insights into the interaction of coronavirus papain-like proteases and interferon-stimulated gene product 15 from different species. J. Mol. Biol. 2017;429(11):1661-1683. DOI 10.1016/j.jmb.2017.04.011.Daczkowski C.M., Dzimianski J.V., Clasman J.R., Goodwin O., Mesecar A.D., Pegan S.D. Structural insights into the interaction of coronavirus papain-like proteases and interferon-stimulated gene product 15 from different species. J. Mol. Biol. 2017;429(11):1661-1683. DOI 10.1016/j.jmb.2017.04.011.Deng X., Baker S.C. An “Old” protein with a new story: Coronavirus endoribonuclease is important for evading host antiviral defenses. Virology. 2018;517:157-163. DOI 10.1016/j.virol.2017.12.024.Deng X., Baker S.C. An “Old” protein with a new story: Coronavirus endoribonuclease is important for evading host antiviral defenses. Virology. 2018;517:157-163. DOI 10.1016/j.virol.2017.12.024.Deng X., Geelen A., Buckley A.C., O’Brien A., Pillatzki A., Lager K.M., Faaberg K.S., Baker S.C. Coronavirus endoribonuclease activity in porcine epidemic diarrhea virus suppresses type I and type III interferon responses. J. Virol. 2019;93(8):e02000-18. DOI 10.1128/JVI.02000-18.Deng X., Geelen A., Buckley A.C., O’Brien A., Pillatzki A., Lager K.M., Faaberg K.S., Baker S.C. Coronavirus endoribonuclease activity in porcine epidemic diarrhea virus suppresses type I and type III interferon responses. J. Virol. 2019;93(8):e02000-18. DOI 10.1128/JVI.02000-18.Egloff M.-P., Ferron F., Campanacci V., Longhi S., Rancurel C., Dutartre H., Snijder E.J., Gorbalenya A.E., Cambillau C., Canard B. The severe acute respiratory syndrome-coronavirus replicative protein nsp9 is a single-stranded RNA-binding subunit unique in the RNA virus world. Proc. Natl. Acad. Sci. USA. 2004;101(11):3792-3796. DOI 10.1073/pnas.0307877101.Egloff M.-P., Ferron F., Campanacci V., Longhi S., Rancurel C., Dutartre H., Snijder E.J., Gorbalenya A.E., Cambillau C., Canard B. The severe acute respiratory syndrome-coronavirus replicative protein nsp9 is a single-stranded RNA-binding subunit unique in the RNA virus world. Proc. Natl. Acad. Sci. USA. 2004;101(11):3792-3796. DOI 10.1073/pnas.0307877101.Frieman M., Ratia K., Johnston R.E., Mesecar A.D., Baric R.S. Severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of IRF3 and NF-kappaB signaling. J. Virol. 2009;83(13):6689-6705. DOI 10.1128/JVI.02220-08.Frieman M., Ratia K., Johnston R.E., Mesecar A.D., Baric R.S. Severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of IRF3 and NF-kappaB signaling. J. Virol. 2009;83(13):6689-6705. DOI 10.1128/JVI.02220-08.Fu L., Ye F., Feng Y., Yu F., Wang Q., Wu Y., Zhao C., Sun H., Huang B., Niu P., Song H., Shi Y., Li X., Tan W., Qi J., Gao G.F. Both Boceprevir and GC376 efficaciously inhibit SARS-CoV-2 by targeting its main protease. Nat. Commun. 2020;11(1):4417. DOI 10.1038/s41467-020-18233-x.Fu L., Ye F., Feng Y., Yu F., Wang Q., Wu Y., Zhao C., Sun H., Huang B., Niu P., Song H., Shi Y., Li X., Tan W., Qi J., Gao G.F. Both Boceprevir and GC376 efficaciously inhibit SARS-CoV-2 by targeting its main protease. Nat. Commun. 2020;11(1):4417. DOI 10.1038/s41467-020-18233-x.Gadhave K., Kumar P., Kumar A., Bhardwaj T., Garg N., Giri R. Conformational dynamics of 13 amino acids long NSP11 of SARS-CoV-2 under membrane mimetics and different solvent conditions. Microb. Pathog. 2021;158:105041. DOI 10.1016/j.micpath.2021.105041.Gadhave K., Kumar P., Kumar A., Bhardwaj T., Garg N., Giri R. Conformational dynamics of 13 amino acids long NSP11 of SARS-CoV-2 under membrane mimetics and different solvent conditions. Microb. Pathog. 2021;158:105041. DOI 10.1016/j.micpath.2021.105041.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. Structure of the RNA-dependent RNA polymerase from COVID-19 virus. Science. 2020;368(6492):779-782. DOI 10.1126/science.abb7498.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. Structure of the RNA-dependent RNA polymerase from COVID-19 virus. Science. 2020;368(6492):779-782. DOI 10.1126/science.abb7498.Goyal B., Goyal D. Targeting the dimerization of the main protease of coronaviruses: a potential broad-spectrum therapeutic strategy. ACS Comb. Sci. 2020;22(6):297-305. DOI 10.1021/acscombsci.0c00058.Goyal B., Goyal D. Targeting the dimerization of the main protease of coronaviruses: a potential broad-spectrum therapeutic strategy. ACS Comb. Sci. 2020;22(6):297-305. DOI 10.1021/acscombsci.0c00058.Graham R.L., Sims A.C., Brockway S.M., Baric R.S., Denison M.R. The nsp2 replicase proteins of murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensable for viral replication. J. Virol. 2005;79(21):13399-13411. DOI 10.1128/JVI.79.21.13399-13411.2005.Graham R.L., Sims A.C., Brockway S.M., Baric R.S., Denison M.R. The nsp2 replicase proteins of murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensable for viral replication. J. Virol. 2005;79(21):13399-13411. DOI 10.1128/JVI.79.21.13399-13411.2005.Hao W., Wojdyla J.A., Zhao R., Han R., Das R., Zlatev I., Manoharan M., Wang M., Cui S. Crystal structure of Middle East respiratory syndrome coronavirus helicase. PLoS Pathog. 2017;13(6):e1006474. DOI 10.1371/journal.ppat.1006474.Hao W., Wojdyla J.A., Zhao R., Han R., Das R., Zlatev I., Manoharan M., Wang M., Cui S. Crystal structure of Middle East respiratory syndrome coronavirus helicase. PLoS Pathog. 2017;13(6):e1006474. DOI 10.1371/journal.ppat.1006474.Hughes S.A., Bonilla P.J., Weiss S.R. Identification of the murine coronavirus p28 cleavage site. J. Virol. 1995;69(2):809-813. DOI 10.1128/JVI.69.2.809-813.1995.Hughes S.A., Bonilla P.J., Weiss S.R. Identification of the murine coronavirus p28 cleavage site. J. Virol. 1995;69(2):809-813. DOI 10.1128/JVI.69.2.809-813.1995.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(20):4933-4942. DOI 10.1038/sj.emboj.7601368.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(20):4933-4942. DOI 10.1038/sj.emboj.7601368.Imbert I., Snijder E.J., Dimitrova M., Guillemot J.-C., Lécine P., Canard B. The SARS-Coronavirus PLnc domain of nsp3 as a replication/transcription scaffolding protein. Virus Res. 2008;133(2):136-148. DOI 10.1016/j.virusres.2007.11.017.Imbert I., Snijder E.J., Dimitrova M., Guillemot J.-C., Lécine P., Canard B. The SARS-Coronavirus PLnc domain of nsp3 as a replication/transcription scaffolding protein. Virus Res. 2008;133(2):136-148. DOI 10.1016/j.virusres.2007.11.017.Ivanov K.A., Thiel V., Dobbe J.C., Meer Y., Snijder E.J., Ziebuhr J. Multiple enzymatic activities associated with severe acute respiratory syndrome coronavirus helicase. J. Virol. 2004;78(11):5619-5632. DOI 10.1128/JVI.78.11.5619-5632.2004.Ivanov K.A., Thiel V., Dobbe J.C., Meer Y., Snijder E.J., Ziebuhr J. Multiple enzymatic activities associated with severe acute respiratory syndrome coronavirus helicase. J. Virol. 2004;78(11):5619-5632. DOI 10.1128/JVI.78.11.5619-5632.2004.Joseph J.S., Saikatendu K.S., Subramanian V., Neuman B.W., Brooun A., Griffith M., Moy K., Yadav M.K., Velasquez J., Buchmeier M.J., Stevens R.C., Kuhn P. Crystal structure of nonstructural protein 10 from the severe acute respiratory syndrome coronavirus reveals a novel fold with two zinc-binding motifs. J. Virol. 2006;80(16): 7894-7901. DOI 10.1128/JVI.00467-06.Joseph J.S., Saikatendu K.S., Subramanian V., Neuman B.W., Brooun A., Griffith M., Moy K., Yadav M.K., Velasquez J., Buchmeier M.J., Stevens R.C., Kuhn P. Crystal structure of nonstructural protein 10 from the severe acute respiratory syndrome coronavirus reveals a novel fold with two zinc-binding motifs. J. Virol. 2006;80(16): 7894-7901. DOI 10.1128/JVI.00467-06.Kamitani W., Narayanan K., Huang C., Lokugamage K., Ikegami T., Ito N., Kubo H., Makino S. Severe acute respiratory syndrome coronavirus nsp1 protein suppresses host gene expression by promoting host mRNA degradation. Proc. Natl. Acad. Sci. USA. 2006; 103(34):12885-12890. DOI 10.1073/pnas.0603144103.Kamitani W., Narayanan K., Huang C., Lokugamage K., Ikegami T., Ito N., Kubo H., Makino S. Severe acute respiratory syndrome coronavirus nsp1 protein suppresses host gene expression by promoting host mRNA degradation. Proc. Natl. Acad. Sci. USA. 2006; 103(34):12885-12890. DOI 10.1073/pnas.0603144103.Kanjanahaluethai A., Baker S.C. Identification of mouse hepatitis virus papain-like proteinase 2 activity. J. Virol. 2000;74(17):7911-7921. DOI 10.1128/jvi.74.17.7911-7921.2000.Kanjanahaluethai A., Baker S.C. Identification of mouse hepatitis virus papain-like proteinase 2 activity. J. Virol. 2000;74(17):7911-7921. DOI 10.1128/jvi.74.17.7911-7921.2000.Kim Y., Jedrzejczak R., Maltseva N.I., Wilamowski M., Endres M., Godzik A., Michalska K., Joachimiak A. Crystal structure of Nsp15 endoribonuclease NendoU from SARS-CoV-2. Protein Sci. 2020; 29(7):1596-1605. DOI 10.1002/pro.3873.Kim Y., Jedrzejczak R., Maltseva N.I., Wilamowski M., Endres M., Godzik A., Michalska K., Joachimiak A. Crystal structure of Nsp15 endoribonuclease NendoU from SARS-CoV-2. Protein Sci. 2020; 29(7):1596-1605. DOI 10.1002/pro.3873.Lu R., Zhao X., Li J., Niu P., Yang B., Wu H., Wang W., Song H., Huang B., Zhu N., Bi Y., Ma X., Zhan F., Wang L., Hu T., Zhou H., Hu Z., Zhou W., Zhao L., Chen J., Meng Y., Wang J., Lin Y., Yuan J., Xie Z., Ma J., Liu W.J., Wang D., Xu W., Holmes E.C., Gao G.F., Wu G., Chen W., Shi W., Tan W. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet. 2020;395(10224):565-574. DOI 10.1016/S0140-6736(20)30251-8.Lu R., Zhao X., Li J., Niu P., Yang B., Wu H., Wang W., Song H., Huang B., Zhu N., Bi Y., Ma X., Zhan F., Wang L., Hu T., Zhou H., Hu Z., Zhou W., Zhao L., Chen J., Meng Y., Wang J., Lin Y., Yuan J., Xie Z., Ma J., Liu W.J., Wang D., Xu W., Holmes E.C., Gao G.F., Wu G., Chen W., Shi W., Tan W. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet. 2020;395(10224):565-574. DOI 10.1016/S0140-6736(20)30251-8.Ma Y., Wu L., Shaw N., Gao Y., Wang J., Sun Y., Lou Z., Yan L., Zhang R., Rao Z. Structural basis and functional analysis of the SARS coronavirus nsp14–nsp10 complex. Proc. Natl. Acad. Sci. USA. 2015;112(30):9436-9441. DOI 10.1073/pnas.1508686112.Ma Y., Wu L., Shaw N., Gao Y., Wang J., Sun Y., Lou Z., Yan L., Zhang R., Rao Z. Structural basis and functional analysis of the SARS coronavirus nsp14–nsp10 complex. Proc. Natl. Acad. Sci. USA. 2015;112(30):9436-9441. DOI 10.1073/pnas.1508686112.Ma-Lauer Y., Carbajo-Lozoya J., Hein M.Y., Müller M.A., Deng W., Lei J., Meyer B., Kusov Y., von Brunn B., Bairad D.R., Hünten S., Drosten C., Hermeking H., Leonhardt H., Mann M., Hilgenfeld R., von Brunn A. p53 down-regulates SARS coronavirus replication and is targeted by the SARS-unique domain and PLpro via E3 ubiquitin ligase RCHY1. Proc. Natl. Acad. Sci. USA. 2016;113(35):E5192-E5201. DOI 10.1073/pnas.1603435113.Ma-Lauer Y., Carbajo-Lozoya J., Hein M.Y., Müller M.A., Deng W., Lei J., Meyer B., Kusov Y., von Brunn B., Bairad D.R., Hünten S., Drosten C., Hermeking H., Leonhardt H., Mann M., Hilgenfeld R., von Brunn A. p53 down-regulates SARS coronavirus replication and is targeted by the SARS-unique domain and PLpro via E3 ubiquitin ligase RCHY1. Proc. Natl. Acad. Sci. USA. 2016;113(35):E5192-E5201. DOI 10.1073/pnas.1603435113.Malik Y.S., Sircar S., Bhat S., Sharun K., Dhama K., Dadar M., Tiwari R., Chaicumpa W. Emerging novel coronavirus (2019-nCoV) – current scenario, evolutionary perspective based on genome analysis and recent developments. Vet. Q. 2020;40(1):68-76. DOI 10.1080/01652176.2020.1727993.Malik Y.S., Sircar S., Bhat S., Sharun K., Dhama K., Dadar M., Tiwari R., Chaicumpa W. Emerging novel coronavirus (2019-nCoV) – current scenario, evolutionary perspective based on genome analysis and recent developments. Vet. Q. 2020;40(1):68-76. DOI 10.1080/01652176.2020.1727993.Miknis Z.J., Donaldson E.F., Umland T.C., Rimmer R.A., Baric R.S., Schultz L.W. Severe acute respiratory syndrome coronavirus nsp9 dimerization is essential for efficient viral growth. J. Virol. 2009; 83(7):3007-3018. DOI 10.1128/JVI.01505-08.Miknis Z.J., Donaldson E.F., Umland T.C., Rimmer R.A., Baric R.S., Schultz L.W. Severe acute respiratory syndrome coronavirus nsp9 dimerization is essential for efficient viral growth. J. Virol. 2009; 83(7):3007-3018. DOI 10.1128/JVI.01505-08.Naqvi A.A.T., Fatima K., Mohammad T., Fatima U., Singh I.K., Singh A., Atif S.M., Hariprasad G., Hasan G.M., Hassan M.I. Insights into SARS-CoV-2 genome, structure, evolution, pathogenesis and therapies: Structural genomics approach. Biochim. Biophys. Acta Mol. Basis Dis. 2020;1866(10):165878. DOI 10.1016/j.bbadis.2020.165878.Naqvi A.A.T., Fatima K., Mohammad T., Fatima U., Singh I.K., Singh A., Atif S.M., Hariprasad G., Hasan G.M., Hassan M.I. Insights into SARS-CoV-2 genome, structure, evolution, pathogenesis and therapies: Structural genomics approach. Biochim. Biophys. Acta Mol. Basis Dis. 2020;1866(10):165878. DOI 10.1016/j.bbadis.2020.165878.Narayanan K., Huang C., Lokugamage K., Kamitani W., Ikegami T., Tseng C.-T.K., Makino S. Severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type I interferon, in infected cells. J. Virol. 2008;82(9):4471-4479. DOI 10.1128/JVI.02472-07.Narayanan K., Huang C., Lokugamage K., Kamitani W., Ikegami T., Tseng C.-T.K., Makino S. Severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type I interferon, in infected cells. J. Virol. 2008;82(9):4471-4479. DOI 10.1128/JVI.02472-07.Ogando N.S., Zevenhoven-Dobbe J.C., Meer Y., Bredenbeek P.J., Posthuma C.C., Snijder E.J. The enzymatic activity of the nsp14 exoribonuclease is critical for replication of MERS-CoV and SARS-CoV-2. J. Virol. 2020;94(23):e01246-20. DOI 10.1128/JVI.01246-20.Ogando N.S., Zevenhoven-Dobbe J.C., Meer Y., Bredenbeek P.J., Posthuma C.C., Snijder E.J. The enzymatic activity of the nsp14 exoribonuclease is critical for replication of MERS-CoV and SARS-CoV-2. J. Virol. 2020;94(23):e01246-20. DOI 10.1128/JVI.01246-20.Oostra M., Hagemeijer M.C., Gent M., Bekker C.P.J., Lintelo E.G., Rottier P.J.M., Haan C.A.M. Topology and membrane anchoring of the coronavirus replication complex: not all hydrophobic domains of nsp3 and nsp6 are membrane spanning. J. Virol. 2008;82(24): 12392-12405. DOI 10.1128/JVI.01219-08.Oostra M., Hagemeijer M.C., Gent M., Bekker C.P.J., Lintelo E.G., Rottier P.J.M., Haan C.A.M. Topology and membrane anchoring of the coronavirus replication complex: not all hydrophobic domains of nsp3 and nsp6 are membrane spanning. J. Virol. 2008;82(24): 12392-12405. DOI 10.1128/JVI.01219-08.Pan J., Peng X., Gao Y., Li Z., Lu X., Chen Y., Ishaq M., Liu D., Dediego M.L., Enjuanes L., Guo D. Genome-wide analysis of proteinprotein interactions and involvement of viral proteins in SARS-CoV replication. PLoS One. 2008;3(10):e3299. DOI 10.1371/journal.pone.0003299.Pan J., Peng X., Gao Y., Li Z., Lu X., Chen Y., Ishaq M., Liu D., Dediego M.L., Enjuanes L., Guo D. Genome-wide analysis of proteinprotein interactions and involvement of viral proteins in SARS-CoV replication. PLoS One. 2008;3(10):e3299. DOI 10.1371/journal.pone.0003299.Pasternak A.O., Spaan W.J.M., Snijder E.J. Nidovirus transcription: how to make sense...? J. Gen. Virol. 2006;87(6):1403-1421. DOI 10.1099/vir.0.81611-0.Pasternak A.O., Spaan W.J.M., Snijder E.J. Nidovirus transcription: how to make sense...? J. Gen. Virol. 2006;87(6):1403-1421. DOI 10.1099/vir.0.81611-0.Rost B. Twilight zone of protein sequence alignments. Protein Eng. 1999;12(2):85-94. DOI 10.1093/protein/12.2.85.Rost B. Twilight zone of protein sequence alignments. Protein Eng. 1999;12(2):85-94. DOI 10.1093/protein/12.2.85.Su D., Lou Z., Sun F., Zhai Y., Yang H., Zhang R., Joachimiak A., Zhang X.C., Bartlam M., Rao1 Z. Dodecamer structure of severe acute respiratory syndrome coronavirus nonstructural protein nsp10. J. Virol. 2006;80(16):7902-7908. DOI 10.1128/JVI.00483-06.Su D., Lou Z., Sun F., Zhai Y., Yang H., Zhang R., Joachimiak A., Zhang X.C., Bartlam M., Rao1 Z. Dodecamer structure of severe acute respiratory syndrome coronavirus nonstructural protein nsp10. J. Virol. 2006;80(16):7902-7908. DOI 10.1128/JVI.00483-06.Subissi L., Posthuma C.C., Collet A., Zevenhoven-Dobbe J.C., Gorbalenya A.E., Decroly E., Snijder E.J., Canard B., Imbert I. One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities. Proc. Natl. Acad. Sci. USA. 2014;111(37):E3900-E3909. DOI 10.1073/pnas.1323705111.Subissi L., Posthuma C.C., Collet A., Zevenhoven-Dobbe J.C., Gorbalenya A.E., Decroly E., Snijder E.J., Canard B., Imbert I. One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities. Proc. Natl. Acad. Sci. USA. 2014;111(37):E3900-E3909. DOI 10.1073/pnas.1323705111.Tahir M. Coronavirus genomic nsp14-ExoN, structure, role, mechanism, and potential application as a drug target. J. Med. Virol. 2021; 93(7):4258-4264. DOI 10.1002/jmv.27009.Tahir M. Coronavirus genomic nsp14-ExoN, structure, role, mechanism, and potential application as a drug target. J. Med. Virol. 2021; 93(7):4258-4264. DOI 10.1002/jmv.27009.Thiel V., Ivanov K.A., Putics Á., Hertzig T., Schelle B., Bayer S., Weißbrich B., Snijder E.J., Rabenau H., Doerr H.W., Gorbalenya A.E., Ziebuhr J. Mechanisms and enzymes involved in SARS coronavirus genome expression. J. Gen. Virol. 2003;84(9):2305-2315. DOI 10.1099/vir.0.19424-0.Thiel V., Ivanov K.A., Putics Á., Hertzig T., Schelle B., Bayer S., Weißbrich B., Snijder E.J., Rabenau H., Doerr H.W., Gorbalenya A.E., Ziebuhr J. Mechanisms and enzymes involved in SARS coronavirus genome expression. J. Gen. Virol. 2003;84(9):2305-2315. DOI 10.1099/vir.0.19424-0.Tomar S., Johnston M.L., John S.E.St., Osswald H.L., Nyalapatla P.R., Paul L.N., Ghosh A.K., Denison M.R., Mesecar A.D. Ligand-induced dimerization of Middle East respiratory syndrome (MERS) coronavirus nsp5 protease (3CLpro): implications for nsp5 regulation and the development of antivirals. J. Biol. Chem. 2015;290(32): 19403-19422. DOI 10.1074/jbc.M115.651463.Tomar S., Johnston M.L., John S.E.St., Osswald H.L., Nyalapatla P.R., Paul L.N., Ghosh A.K., Denison M.R., Mesecar A.D. Ligand-induced dimerization of Middle East respiratory syndrome (MERS) coronavirus nsp5 protease (3CLpro): implications for nsp5 regulation and the development of antivirals. J. Biol. Chem. 2015;290(32): 19403-19422. DOI 10.1074/jbc.M115.651463.Velthuis A.J.W., Arnold J.J., Cameron C.E., Worm S.H.E., Snijder E.J. The RNA polymerase activity of SARS-coronavirus nsp12 is primer dependent. Nucleic Acids Res. 2010;38(1):203-214. DOI 10.1093/nar/gkp904.Velthuis A.J.W., Arnold J.J., Cameron C.E., Worm S.H.E., Snijder E.J. The RNA polymerase activity of SARS-coronavirus nsp12 is primer dependent. Nucleic Acids Res. 2010;38(1):203-214. DOI 10.1093/nar/gkp904.Velthuis A.J.W., Worm S.H.E., Snijder E.J. The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension. Nucleic Acids Res. 2012;40(4):1737-1747. DOI 10.1093/nar/gkr893.Velthuis A.J.W., Worm S.H.E., Snijder E.J. The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension. Nucleic Acids Res. 2012;40(4):1737-1747. DOI 10.1093/nar/gkr893.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(8):4444-4454. DOI 10.1128/JVI.06635-11.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(8):4444-4454. DOI 10.1128/JVI.06635-11.Zeng Z., Deng F., Shi K., Ye G., Wang G., Fang L., Xiao S., Fu Z., Peng G. Dimerization of coronavirus nsp9 with diverse modes enhances its nucleic acid binding affinity. J. Virol. 2018;92(17): e00692-18. DOI 10.1128/JVI.00692-18.Zeng Z., Deng F., Shi K., Ye G., Wang G., Fang L., Xiao S., Fu Z., Peng G. Dimerization of coronavirus nsp9 with diverse modes enhances its nucleic acid binding affinity. J. Virol. 2018;92(17): e00692-18. DOI 10.1128/JVI.00692-18.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(11):980-986. DOI 10.1038/nsmb999.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(11):980-986. DOI 10.1038/nsmb999.Zhang L., Li L., Yan L., Ming Z., Jia Z., Lou Z., Rao Z. Structural and biochemical characterization of endoribonuclease nsp15 encoded by Middle East respiratory syndrome coronavirus. J. Virol. 2018; 92(22):e00893-18. DOI 10.1128/JVI.00893-18.Zhang L., Li L., Yan L., Ming Z., Jia Z., Lou Z., Rao Z. Structural and biochemical characterization of endoribonuclease nsp15 encoded by Middle East respiratory syndrome coronavirus. J. Virol. 2018; 92(22):e00893-18. DOI 10.1128/JVI.00893-18.Ziebuhr J., Snijder E.J., Gorbalenya A.E. Virus-encoded proteinases and proteolytic processing in the Nidovirales. J. Gen. Virol. 2000; 81(4):853-879. DOI 10.1099/0022-1317-81-4-853.Ziebuhr J., Snijder E.J., Gorbalenya A.E. Virus-encoded proteinases and proteolytic processing in the Nidovirales. J. Gen. Virol. 2000; 81(4):853-879. DOI 10.1099/0022-1317-81-4-853.

The authors declare that there are no conflicts of interest present.