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Biochem Biophys Res Commun. 2019 Dec 10;520(3):499-506. doi: 10.1016/j.bbrc.2019.09.115. Epub 2019 Oct 5.

An in vivo cell-based assay for investigating the specific interaction between the SARS-CoV N-protein and its viral RNA packaging sequence.

Author information

1
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.
2
Columbia University Vagelos College of Physicians and Surgeons, New York, NY, 10032, USA.
3
Division of Nephrology, Department of Internal Medicine, Gangnam Severance Hospital, Yonsei University, Seoul, 06273, Republic of Korea.
4
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. Electronic address: yecho@yuhs.ac.

Abstract

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.

KEYWORDS:

Cell-based assay; Coronavirus; Nucleocapsid; Packaging signal sequence; Protein-RNA interaction; SARS

PMID:
31594639
DOI:
10.1016/j.bbrc.2019.09.115

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