Autor(es): Zimmermann, Liv ; Zhao, Xiaohan ; Makroczyova, Jana ; Wachsmuth-Melm, Moritz ; Prasad, Vibhu ; Hensel, Zach ; Bartenschlager, Ralf ; Chlanda, Petr
Data: 2023
Identificador Persistente: http://hdl.handle.net/10362/165728
Origem: Repositório Institucional da UNL
Assunto(s): Chemistry(all); Biochemistry, Genetics and Molecular Biology(all); Physics and Astronomy(all)
Funding Information: We thank the Infectious Diseases Imaging Platform (IDIP) at the Center for Integrative Infectious Disease Research Heidelberg, the cryo-EM network at the Heidelberg University (HD-cryoNET) and Heidelberg University Electron Microscopy Core Facility for support and assistance. The authors gratefully acknowledge the data storage service SDS@hd supported by the Ministry of Science, Research, and the Arts Baden-Württemberg (MWK), the German Research Foundation (DFG) through grant INST 35/1314-1 FUGG and INST 35/1503-1 FUGG. This work was supported by a research grant from the Chica and Heinz Schaller Foundation (Schaller Research Group Leader Program). Work of P.C. and R.B. is supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) project no. 240245660–SFB1129 and project number 437060729 (PC and MWM). In addition, work of R.B. is supported by DFG project no. 272983813–TRR 179) and by the project “Virological and immunological determinants of COVID-19 pathogenesis – lessons to get prepared for future pandemics (KA1-Co-02 “COVIPA”)”, a grant from the Helmholtz Association’s Initiative and Networking Fund. V.P. is supported by a European Molecular Biology Organization (EMBO) long-term fellowship (ALTF454-2020). LZ and JM are supported by CoVLP project of the Flagship Initiative Engineering Molecular Systems. ZH received support for this work from FCT - Fundação para a Ciência e a Tecnologia, I.P., through MOSTMICRO-ITQB R&D Unit (UIDB/04612/2020, 510 UIDP/04612/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020) and from a joint research agreement with the Okinawa Institute of Science and Technology. Funding Information: We thank the Infectious Diseases Imaging Platform (IDIP) at the Center for Integrative Infectious Disease Research Heidelberg, the cryo-EM network at the Heidelberg University (HD-cryoNET) and Heidelberg University Electron Microscopy Core Facility for support and assistance. The authors gratefully acknowledge the data storage service SDS@hd supported by the Ministry of Science, Research, and the Arts Baden-Württemberg (MWK), the German Research Foundation (DFG) through grant INST 35/1314-1 FUGG and INST 35/1503-1 FUGG. This work was supported by a research grant from the Chica and Heinz Schaller Foundation (Schaller Research Group Leader Program). Work of P.C. and R.B. is supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) project no. 240245660–SFB1129 and project number 437060729 (PC and MWM). In addition, work of R.B. is supported by DFG project no. 272983813–TRR 179) and by the project “Virological and immunological determinants of COVID-19 pathogenesis – lessons to get prepared for future pandemics (KA1-Co-02 “COVIPA”)”, a grant from the Helmholtz Association’s Initiative and Networking Fund. V.P. is supported by a European Molecular Biology Organization (EMBO) long-term fellowship (ALTF454-2020). LZ and JM are supported by CoVLP project of the Flagship Initiative Engineering Molecular Systems. ZH received support for this work from FCT - Fundação para a Ciência e a Tecnologia, I.P., through MOSTMICRO-ITQB R&D Unit (UIDB/04612/2020, 510 UIDP/04612/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020) and from a joint research agreement with the Okinawa Institute of Science and Technology. Publisher Copyright: © 2023, The Author(s).
Coronavirus replication is associated with the remodeling of cellular membranes, resulting in the formation of double-membrane vesicles (DMVs). A DMV-spanning pore was identified as a putative portal for viral RNA. However, the exact components and the structure of the SARS-CoV-2 DMV pore remain to be determined. Here, we investigate the structure of the DMV pore by in situ cryo-electron tomography combined with subtomogram averaging. We identify non-structural protein (nsp) 3 and 4 as minimal components required for the formation of a DMV-spanning pore, which is dependent on nsp3-4 proteolytic cleavage. In addition, we show that Mac2-Mac3-DPUP-Ubl2 domains are critical for nsp3 oligomerization and crown integrity which influences membrane curvature required for biogenesis of DMVs. Altogether, SARS-CoV-2 nsp3-4 have a dual role by driving the biogenesis of replication organelles and assembly of DMV-spanning pores which we propose here to term replicopores.
