Coronaviruses remodel the intracellular host membranes during replication, forming double-membrane vesicles (DMVs) to accommodate viral RNA synthesis and modifications^1,2. 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^3,4,5. 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.

冠状病毒在复制过程中重塑细胞内宿主膜，形成双膜囊泡 (DMV) 以适应病毒 RNA 合成和修饰^1,2 。 SARS-CoV-2 非结构蛋白 3 (nsp3) 和 nsp4 是诱导 DMV 形成和形成双膜跨孔所需的最小病毒成分，对于运输新合成的病毒 RNA 至关重要^3,4,5 。 DMV 孔复合物形成的机制仍不清楚。在这里，我们描述了 SARS-CoV-2 nsp3-nsp4 孔复合物的分子结构，通过低温电子断层扫描和分离的 DMV 中的亚断层图平均来解析。这些结构揭示了 nsp3-nsp4 孔复合物的意想不到的化学计量和拓扑结构，该复合物包含 nsp3 和 nsp4 各 12 个副本，组织成 4 个同心堆叠六聚体环，模仿微型核孔复合物。跨膜域相互交叉，在双膜连接处产生高局部曲率，将双膜重组与孔形成耦合起来。胞外域以伪 12 重对称性形成广泛的接触，将孔复合物从膜间空间带出。中央带正电荷的精氨酸残基环协调假定的 RNA 易位，这对于病毒复制至关重要。我们的工作建立了一个了解 DMV 孔形成和 RNA 易位的框架，为开发新的抗病毒策略来对抗冠状病毒感染提供了结构基础。