Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul, 03722, Republic of Korea; The Spine and Spinal Cord Institute, Department of Neurosurgery, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, 06273, Republic of Korea.
Columbia University Vagelos College of Physicians and Surgeons, New York, NY, 10032, USA.
Biochem Biophys Res Commun. 2019 Dec 10;520(3):499-506. doi: 10.1016/j.bbrc.2019.09.115. Epub 2019 Oct 5.
The SARS-CoV nucleocapsid (N) protein serves multiple functions in viral replication, transcription, and assembly of the viral genome complex. Coronaviruses specifically package genomic RNA into assembled virions, and in SARS-CoV, it is reported that this process is driven by an interaction between the N-protein and a packaging signal encoded within the viral RNA. While recent studies have uncovered the sequence of this packaging signal, little is known about the specific interaction between the N-protein and the packaging signal sequence, and the mechanisms by which this interaction drives viral genome packaging. In this study, we developed a novel in vivo cell-based assay for examining this interaction between the N-protein and packaging signal RNA for SARS-CoV, as well as other viruses within the coronaviridae family. Our results demonstrate that the N-protein specifically recognizes the SARS-CoV packaging signal with greater affinity compared to signals from other coronaviruses or non-coronavirus species. We also use deletion mapping to identify a 151-nt region within the packaging signal sequence that is critical for N-protein-RNA binding, and conversely, we show that both the N-terminal and C-terminal domains of the N protein are necessary for recognizing the packaging RNA. These results describe, for the first time, in vivo evidence for an interaction between the SARS-CoV N-protein and its packaging signal RNA, and demonstrate the feasibility of using this cell-based assay to further probe viral RNA-protein interactions in future studies.
SARS-CoV 的核衣壳（N）蛋白在病毒复制、转录和基因组复合物的组装中具有多种功能。冠状病毒专门将基因组 RNA 包装到组装的病毒粒子中，在 SARS-CoV 中，据报道，这个过程是由 N 蛋白与病毒 RNA 内编码的包装信号之间的相互作用驱动的。虽然最近的研究已经揭示了这个包装信号的序列，但人们对 N 蛋白与包装信号序列之间的具体相互作用以及这种相互作用驱动病毒基因组包装的机制知之甚少。在这项研究中，我们开发了一种新的基于细胞的体内测定法，用于研究 SARS-CoV 及其冠状病毒科内其他病毒的 N 蛋白与包装信号 RNA 之间的相互作用。我们的结果表明，N 蛋白特异性地识别 SARS-CoV 包装信号，与其他冠状病毒或非冠状病毒的信号相比，亲和力更高。我们还使用缺失作图来鉴定包装信号序列内的 151 个核苷酸区域，该区域对于 N 蛋白-RNA 结合至关重要，反过来，我们表明 N 蛋白的 N 端和 C 端结构域对于识别包装 RNA 都是必需的。这些结果首次在体内描述了 SARS-CoV N 蛋白与其包装信号 RNA 之间的相互作用，并证明了使用这种基于细胞的测定法在未来研究中进一步探测病毒 RNA-蛋白相互作用的可行性。
