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TitleSARS-CoV-2 nsp3 and nsp4 are minimal constituents of a pore spanning replication organelle.
Journal, issue, pagesNat Commun, Vol. 14, Issue 1, Page 7894, Year 2023
Publish dateNov 30, 2023
AuthorsLiv Zimmermann / Xiaohan Zhao / Jana Makroczyova / Moritz Wachsmuth-Melm / Vibhu Prasad / Zach Hensel / Ralf Bartenschlager / Petr Chlanda /
PubMed AbstractCoronavirus 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 ...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.
External linksNat Commun / PubMed:38036567 / PubMed Central
MethodsEM (tomography) / EM (subtomogram averaging)
Resolution18.0 - 30.0 Å
Structure data

EMDB-15925: Cryo-electron tomogram acquired on cryo-lamella of VeroE6 cells transfected with SARS-CoV-2 HA-nsp3-4-V5, plunge frozen at 16 hpt
Method: EM (tomography)

EMDB-15926: Cryo-electron tomogram of VeroE6 cells transfected with HA-nsp3-deltaUbl1-Ubl2-nsp4-V5, plunge frozen at 16 hpt
Method: EM (tomography)

EMDB-15927: Cryo-electron tomogram of VeroE6 cells transfected with HA-nsp3-deltaUbl1-Mac1-nsp4-V5. Cells were plunge-frozen at 16h post-transfection and subjected to cryo-FIB milling.
Method: EM (tomography)

EMDB-15928: Cryo-electron tomogram of VeroE6 cells transfected with HA-GG>AA-nsp4-V5, plunge-frozen at 16 hpt and subjected to cryo-FIB milling.
Method: EM (tomography)

EMDB-15929: Cryo-electron tomogram of VeroE6 cells transfected with nsp3-4, vitrified by plunge-freezing at 16 hours post transfection and processed by cryo-FIB milling.
Method: EM (tomography)

EMDB-15963: Subtomogram average SARS-CoV-2 nsp3-4 from cryo-ET of VeroE6 expressing nsp3-4 (filtered to 20A resolution).
Method: EM (subtomogram averaging) / Resolution: 20.3 Å

EMDB-15964: Subtomogram averaging of SARS-CoV-2 nsp3-4 delta Ubl1-Mac1 obtained from cryo-ET of cryo-FIB milled VeroE6 cells transfected with nsp3-4 delta Ubl1-Mac1
Method: EM (subtomogram averaging) / Resolution: 18.0 Å

EMDB-15965: Subtomogram average of SARS-CoV-2 nsp3-4 delate Ubl1-Ubl2 obtained from cryo-ET of cryo-FIB milled VeroE6 cells transfected with nsp3-4 delta Ubl1-Ubl2
Method: EM (subtomogram averaging) / Resolution: 30.0 Å

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  • Severe acute respiratory syndrome coronavirus 2

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