Replication Data for: Assembly of SARS-CoV-2 nucleocapsid protein with nucleic acid

The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-)protein into ribonucleoprotein particles (RNPs), 38±10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining mutant proteins in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multivalent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N-protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的病毒基因组被核衣壳（nucleocapsid, N-）蛋白包装为核糖核蛋白颗粒（ribonucleoprotein particles, RNPs），每个病毒粒子（virion）中包含38±10个此类颗粒。由于RNPs本身具有多形性、N蛋白内在无序区域的高度柔性，以及病毒RNA与N蛋白N端结构域（N-terminal domain, NTD）和C端结构域（C-terminal domain, CTD）内结合位点间存在高度多价相互作用，其结构一直未能明确。本研究结合多种生物物理技术，通过体外RNP形成实验检测结合不同核酸的突变蛋白，并在病毒组装实验中对突变蛋白进行分析，以此探究RNPs的关键相互作用基序。研究发现，结合核酸的N蛋白二聚体通过新近报道的蛋白-蛋白相互作用界面发生寡聚化，该界面由其NTD与CTD之间较长的无序连接区域内的瞬时螺旋所介导。由此形成的六聚体复合物通过多价蛋白-核酸相互作用得以稳定，该相互作用可在二聚体亚基之间建立交联。N蛋白的二聚体C端结构域可提供一个以上结合茎环RNA（stem-loop RNA）的位点，进一步稳定组装体。本研究提出了一种RNP组装模型：病毒RNA表面的高密度N蛋白支架先形成，随后通过无序连接区域内的蛋白-蛋白相互作用发生协同多聚化。