Structural basis of RNA recognition by the SARS-CoV-2 nucleocapsid phosphoprotein.

Dhurvas Chandrasekaran Dinesh,Radim Nencka,Dominika Chalupska,Jan Silhan,Evzen Boura,Eliska Koutna,Vaclav Veverka,Michael S Diamond

doi:10.1371/journal.ppat.1009100

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19). SARS-CoV-2 is a single-stranded positive-sense RNA virus. Like other coronaviruses, SARS-CoV-2 has an unusually large genome that encodes four structural proteins and sixteen nonstructural proteins. The structural nucleocapsid phosphoprotein N is essential for linking the viral genome to the viral membrane. Both N-terminal RNA binding (N-NTD) and C-terminal dimerization domains are involved in capturing the RNA genome and, the intrinsically disordered region between these domains anchors the ribonucleoprotein complex to the viral membrane. Here, we characterized the structure of the N-NTD and its interaction with RNA using NMR spectroscopy. We observed a positively charged canyon on the surface of the N-NTD that might serve as a putative RNA binding site similarly to other coronaviruses. The subsequent NMR titrations using single-stranded and double-stranded RNA revealed a much more extensive U-shaped RNA-binding cleft lined with regularly distributed arginines and lysines. The NMR data supported by mutational analysis allowed us to construct hybrid atomic models of the N-NTD/RNA complex that provided detailed insight into RNA recognition.

Highlights

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) of the Coronaviridae family [1]
We describe how the N protein binds single- and double-stranded RNA, a key process for virion assembly
The CTD serves as a dimerization domain and the intrinsically disordered region (IDR) between the domains interact with the matrix protein forming the physical link between the +RNA genome and envelope

Summary

Introduction

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) of the Coronaviridae family [1]. The fourth structural protein, the nucleocapsid phosphoprotein (N), physically links the envelope to the +RNA genome, interacts with the endodomain of the viral membrane protein M [6] and plays a central role in the packaging signal RNA recognition and subsequent RNA encapsidation [7,8]. It consists of an N-terminal (NTD) and a C-terminal (CTD) domain (Fig 1) that are both capable of RNA binding. In light of the genomic similarities between SARS-CoV and SARS-CoV-2, it is reasonable to expect the SARS-CoV-2 N protein to function analogously

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