Molecular architecture of coronavirus double-membrane vesicle pore complex

Abstract

<p>Coronaviruses remodel the intracellular host membranes during replication, forming double-membrane vesicles (DMVs) to accommodate viral RNA synthesis and modifications^{<a title="Wolff, G., Melia, C. E., Snijder, E. J. & Barcena, M. Double-membrane vesicles as platforms for viral replication. Trends Microbiol. 28, 1022–1033 (2020)." href="https://www.nature.com/articles/s41586-024-07817-y#ref-CR1">1</a>,<a title="Yan, W., Zheng, Y., Zeng, X., He, B. & Cheng, W. Structural biology of SARS-CoV-2: open the door for novel therapies. Signal Transduct. Target. Ther. 7, 26 (2022)." href="https://www.nature.com/articles/s41586-024-07817-y#ref-CR2">2</a>}. SARS-CoV-2 non-structural protein 3 (nsp3) and nsp4 are the minimal viral components required to induce DMV formation and to form a double-membrane-spanning pore, essential for the transport of newly synthesized viral RNAs^{<a title="Wolff, G. et al. A molecular pore spans the double membrane of the coronavirus replication organelle. Science 369, 1395–1398 (2020)." href="https://www.nature.com/articles/s41586-024-07817-y#ref-CR3">3</a>,<a title="Mendonca, L. et al. Correlative multi-scale cryo-imaging unveils SARS-CoV-2 assembly and egress. Nat. Commun. 12, 4629 (2021)." href="https://www.nature.com/articles/s41586-024-07817-y#ref-CR4">4</a>,<a title="Wolff, G. & Barcena, M. Multiscale electron microscopy for the study of viral replication organelles. Viruses 13, 197 (2021)." href="https://www.nature.com/articles/s41586-024-07817-y#ref-CR5">5</a>}. The mechanism of DMV pore complex formation remains unknown. Here we describe the molecular architecture of the SARS-CoV-2 nsp3–nsp4 pore complex, as resolved by cryogenic electron tomography and subtomogram averaging in isolated DMVs. The structures uncover an unexpected stoichiometry and topology of the nsp3–nsp4 pore complex comprising 12 copies each of nsp3 and nsp4, organized in 4 concentric stacking hexamer rings, mimicking a miniature nuclear pore complex. The transmembrane domains are interdigitated to create a high local curvature at the double-membrane junction, coupling double-membrane reorganization with pore formation. The ectodomains form extensive contacts in a pseudo-12-fold symmetry, belting the pore complex from the intermembrane space. A central positively charged ring of arginine residues coordinates the putative RNA translocation, essential for virus replication. Our work establishes a framework for understanding DMV pore formation and RNA translocation, providing a structural basis for the development of new antiviral strategies to combat coronavirus infection.<br></p>