The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a β-coronavirus, is the causative agent of the COVID-19 pandemic. Like for other coronaviruses, its particles are composed of four structural proteins: Spike (S), Envelope (E), Membrane (M) and Nucleoprotein (N) proteins. The involvement of each of these proteins and their interactions are critical for assembly and production of β-coronavirus particles. Here, we sought to characterize the interplay of SARS-CoV-2 structural proteins during the viral assembly process. By combining biochemical and imaging assays in infected vs. transfected cells, we show that E and M regulate intracellular trafficking of S as well as its intracellular processing. Indeed, the imaging data reveal that S is re-localized at endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) or Golgi compartments upon co-expression of E or M, as observed in SARS-CoV-2-infected cells, which prevents syncytia formation. We show that a C-terminal retrieval motif in the cytoplasmic tail of S is required for its M-mediated retention in the ERGIC, whereas E induces S retention by modulating the cell secretory pathway. We also highlight that E and M induce a specific maturation of N-glycosylation of S, independently of the regulation of its localization, with a profile that is observed both in infected cells and in purified viral particles. Finally, we show that E, M and N are required for optimal production of virus- like-particles. Altogether, these results highlight how E and M proteins may influence the properties of S proteins and promote the assembly of SARS-CoV-2 viral particles.

中文翻译：

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SARS-CoV-2 包膜蛋白和膜蛋白调节刺突蛋白的成熟和保留，从而允许病毒样颗粒的组装


严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 是一种 β 冠状病毒，是 COVID-19 大流行的病原体。与其他冠状病毒一样，其颗粒由四种结构蛋白组成：刺突蛋白 (S)、包膜蛋白 (E)、膜蛋白 (M) 和核蛋白 (N)。这些蛋白质的参与及其相互作用对于β-冠状病毒颗粒的组装和生产至关重要。在这里，我们试图表征 SARS-CoV-2 结构蛋白在病毒组装过程中的相互作用。通过结合感染细胞和转染细胞的生化和成像检测，我们发现 E 和 M 调节 S 的细胞内运输及其细胞内加工。事实上，成像数据显示，正如在 SARS-CoV-2 感染细胞中观察到的那样，在 E 或 M 共表达后，S 重新定位于内质网 (ER)-高尔基体中间区室 (ERGIC) 或高尔基体区室，这防止合胞体形成。我们发现，S 细胞质尾部的 C 端修复基序是 M 介导的 ERGIC 保留所必需的，而 E 通过调节细胞分泌途径诱导 S 保留。我们还强调，E 和 M 诱导 S 的 N-糖基化的特异性成熟，独立于其定位的调节，其特征在受感染的细胞和纯化的病毒颗粒中都观察到。最后，我们证明 E、M 和 N 是病毒样颗粒最佳生产所必需的。总而言之，这些结果强调了 E 和 M 蛋白如何影响 S 蛋白的特性并促进 SARS-CoV-2 病毒颗粒的组装。