Wang Yan, Ling Xiaobin, Zhang Chong, Zou Jian, Luo Bingnan, Luo Yongbo, Jia Xinyu, Jia Guowen, Zhang Minghua, Hu Junchao, Liu Ting, Wang Yuanfeiyi, Lu Kefeng, Li Dan, Ma Jinbiao, Liu Cong, Su Zhaoming
The State Key Laboratory of Biotherapy, Frontiers Medical Center of Tianfu Jincheng Laboratory, National Clinical Research Center for Geriatrics and Department of Geriatrics, West China Hospital, Sichuan University, Chengdu, 610044, Sichuan, China.
State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, Department of Biochemistry and Biophysics, School of Life Sciences, Fudan University, Shanghai, 200438, China.
Mol Biomed. 2023 May 22;4(1):16. doi: 10.1186/s43556-023-00129-z.
SARS-CoV-2 and its variants, with the Omicron subvariant XBB currently prevailing the global infections, continue to pose threats on public health worldwide. This non-segmented positive-stranded RNA virus encodes the multi-functional nucleocapsid protein (N) that plays key roles in viral infection, replication, genome packaging and budding. N protein consists of two structural domains, NTD and CTD, and three intrinsically disordered regions (IDRs) including the N, the serine/arginine rich motif (SR), and the C. Previous studies revealed functions of N protein in RNA binding, oligomerization, and liquid-liquid phase separation (LLPS), however, characterizations of individual domains and their dissected contributions to N protein functions remain incomplete. In particular, little is known about N protein assembly that may play essential roles in viral replication and genome packing. Here, we present a modular approach to dissect functional roles of individual domains in SARS-CoV-2 N protein that reveals inhibitory or augmented modulations of protein assembly and LLPS in the presence of viral RNAs. Intriguingly, full-length N protein (N) assembles into ring-like architecture whereas the truncated SR-CTD-C (N) promotes filamentous assembly. Moreover, LLPS droplets of N and N are significantly enlarged in the presence of viral RNAs, and we observed filamentous structures in the N droplets using correlative light and electron microscopy (CLEM), suggesting that the formation of LLPS droplets may promote higher-order assembly of N protein for transcription, replication and packaging. Together this study expands our understanding of the multiple functions of N protein in SARS-CoV-2.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）及其变体，目前奥密克戎亚型XBB在全球感染中占主导地位，继续对全球公共卫生构成威胁。这种非节段性正链RNA病毒编码多功能核衣壳蛋白（N），该蛋白在病毒感染、复制、基因组包装和出芽过程中发挥关键作用。N蛋白由两个结构域，即N端结构域（NTD）和C端结构域（CTD），以及三个内在无序区域（IDR）组成，包括N区、富含丝氨酸/精氨酸的基序（SR）和C区。先前的研究揭示了N蛋白在RNA结合、寡聚化和液-液相分离（LLPS）方面的功能，然而，各个结构域的特性及其对N蛋白功能的具体贡献仍不完整。特别是，关于可能在病毒复制和基因组包装中起重要作用的N蛋白组装知之甚少。在这里，我们提出了一种模块化方法来剖析SARS-CoV-2 N蛋白中各个结构域的功能作用，该方法揭示了在病毒RNA存在下对蛋白质组装和LLPS的抑制或增强调节。有趣的是，全长N蛋白（N）组装成环状结构，而截短的SR-CTD-C（N）促进丝状组装。此外，在病毒RNA存在下，N和N的LLPS液滴显著增大，并且我们使用相关光电子显微镜（CLEM）在N液滴中观察到丝状结构，这表明LLPS液滴的形成可能促进N蛋白的高阶组装以进行转录、复制和包装。这项研究共同扩展了我们对SARS-CoV-2中N蛋白多种功能的理解。
