Coronaviruses infect many different species including humans. The last two decades have seen three zoonotic coronaviruses, with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) causing a pandemic in 2020. Coronaviral non-structural proteins (nsps) form the replication-transcription complex (RTC). Nsp7 and nsp8 interact with and regulate the RNA-dependent RNA-polymerase and other enzymes in the RTC. However, the structural plasticity of nsp7+8 complexes has been under debate. Here, we present the framework of nsp7+8 complex stoichiometry and topology based on native mass spectrometry and complementary biophysical techniques of nsp7+8 complexes from seven coronaviruses in the genera Alpha- and Betacoronavirus including SARS-CoV-2. Their complexes cluster into three groups, which systematically form either heterotrimers or heterotetramers or both, exhibiting distinct topologies. Moreover, even at high protein concentrations, SARS-CoV-2 nsp7+8 consists primarily of heterotetramers. From these results, the different assembly paths can be pinpointed to specific residues and an assembly model proposed.

冠状病毒可感染包括人类在内的许多不同物种。在过去的二十年中，出现了三种人畜共患型冠状病毒，其中SARS-CoV-2（严重急性呼吸系统综合症冠状病毒2）在2020年引起大流行。冠状病毒非结构蛋白（nsps）形成了复制转录复合物（RTC）。Nsp7和nsp8与RTC中的RNA依赖性RNA聚合酶和其他酶相互作用并调节它们。但是，nsp7 + 8配合物的结构可塑性一直在争论中。这里，我们提出nsp7的+ 8复杂化学计量和拓扑的框架基于天然质谱和来自七个冠状nsp7的+ 8种配合物的互补生物物理技术在属甲型和乙型包括SARS-CoV-2。它们的复合物分为三类，系统地形成异三聚体或异四聚体或两者，表现出独特的拓扑结构。而且，即使在高蛋白浓度下，SARS-CoV-2 nsp7 + 8仍主要由异四聚体组成。根据这些结果，可以将不同的组装路径精确定位到特定的残基，并提出组装模型。