The SARS-CoV-2 nucleocapsid (N) protein is a highly immunogenic viral protein that plays essential roles in replication and virion assembly. Here, using native mass spectrometry, we show that dimers are the functional unit of ribonucleoprotein assembly and that N protein binds RNA with a preference for GGG motifs, a common motif in coronavirus packaging signals. Unexpectedly, proteolytic processing of N protein resulted in the formation of additional proteoforms. The N-terminal proteoforms bind RNA, with the same preference for GGG motifs, and bind to cyclophilin A, an interaction which can be abolished by approved immunosuppressant cyclosporin A. Furthermore, N proteoforms showed significantly different interactions with IgM, IgG, and IgA antibodies from convalescent plasma. Notably, the C-terminal proteoform exhibited a heightened interaction with convalescent antibodies, suggesting the antigenic epitope is localized to the C-terminus. Overall, the different interactions of N proteoforms highlight potential avenues for therapeutic intervention and identify a stable and immunogenic proteoform as a possible candidate for immune-directed therapies.

中文翻译：

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SARS-CoV-2 N 蛋白与 RNA、宿主蛋白和恢复期抗体的蛋白质特异性相互作用促进其发挥多种作用

SARS-CoV-2 核衣壳 (N) 蛋白是一种高度免疫原性的病毒蛋白，在复制和病毒粒子组装中发挥重要作用。在这里，我们使用天然质谱法，证明二聚体是核糖核蛋白组装的功能单位，并且 N 蛋白优先结合 RNA，并优先选择 GGG 基序，这是冠状病毒包装信号中的常见基序。出乎意料的是，N 蛋白的蛋白水解加工导致了额外蛋白质形式的形成。N 端蛋白质形式与 RNA 结合，对 GGG 基序具有相同的偏好，并与亲环蛋白 A 结合，这种相互作用可以被批准的免疫抑制剂环孢菌素 A 消除。此外，N 蛋白质形式与 IgM、IgG 和 IgA 抗体的相互作用表现出显着不同的相互作用来自恢复期血浆。值得注意的是，C 端蛋白质形式与恢复期抗体的相互作用增强，表明抗原表位位于 C 端。总体而言，N 蛋白质形式的不同相互作用突出了治疗干预的潜在途径，并确定了稳定且具有免疫原性的蛋白质形式作为免疫导向疗法的可能候选者。