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Structural dynamics of SARS-CoV-2 nucleocapsid protein induced by RNA binding

['Helder Veras Ribeiro-Filho', 'Brazilian Biosciences National Laboratory', 'Brazilian Center For Research In Energy', 'Materials', 'Cnpem', 'Campinas', 'Gabriel Ernesto Jara', 'Fernanda Aparecida Heleno Batista', 'Gabriel Ravanhani Schleder', 'Brazilian Nanotechnology National Laboratory']

Date: 2022-06

The nucleocapsid (N) protein of the SARS-CoV-2 virus, the causal agent of COVID-19, is a multifunction phosphoprotein that plays critical roles in the virus life cycle, including transcription and packaging of the viral RNA. To play such diverse roles, the N protein has two globular RNA-binding modules, the N- (NTD) and C-terminal (CTD) domains, which are connected by an intrinsically disordered region. Despite the wealth of structural data available for the isolated NTD and CTD, how these domains are arranged in the full-length protein and how the oligomerization of N influences its RNA-binding activity remains largely unclear. Herein, using experimental data from electron microscopy and biochemical/biophysical techniques combined with molecular modeling and molecular dynamics simulations, we show that, in the absence of RNA, the N protein formed structurally dynamic dimers, with the NTD and CTD arranged in extended conformations. However, in the presence of RNA, the N protein assumed a more compact conformation where the NTD and CTD are packed together. We also provided an octameric model for the full-length N bound to RNA that is consistent with electron microscopy images of the N protein in the presence of RNA. Together, our results shed new light on the dynamics and higher-order oligomeric structure of this versatile protein.

The nucleocapsid (N) protein of the SARS-CoV-2 virus plays an essential role in virus particle assembly as it specifically binds to and wraps the virus genomic RNA into a well-organized structure known as the ribonucleoprotein. Understanding how the N protein wraps around the virus RNA is critical for the development of strategies to inhibit virus assembly within host cells. One of the limitations regarding the molecular structure of the ribonucleoprotein, however, is that the N protein has several unstructured and mobile regions that preclude the resolution of its full atomic structure. Moreover, the N protein can form higher-order oligomers, both in the presence and absence of RNA. Here we employed computational methods, supported by experimental data, to simulate the N protein structural dynamics in the absence and presence of RNA. Our data suggest that the N protein forms structurally dynamic dimers in the absence of RNA, with its structured N- and C-terminal domains oriented in extended conformations. In the presence of RNA, however, the N protein assumes a more compact conformation. Our model for the oligomeric structure of the N protein bound to RNA helps to understand how N protein dimers interact to each other to form the ribonucleoprotein.

Funding: This work is part of the Rede Virus MCTI taskforce on COVID-19 funded by FINEP (grant number 01.20.0003.00) ( http://www.finep.gov.br/ ), Brazilian Ministry of Science, Technology and Innovation ( https://www.gov.br/mcti/ ). GRS received financial support from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP - https://fapesp.br/ ), project number 17/18139-6. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: All data related to the computational simulations are available from Zenodo repository (DOI: 10.5281/zenodo.5841603 ). All other data needed to evaluate the conclusions in the paper are present in the main manuscript and the Supplementary Materials.

Copyright: © 2022 Ribeiro-Filho et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Due to its dynamic structure and multiple oligomeric organization depending on environmental conditions, and to the fact that the N protein is also modified by phosphorylation [ 28 , 29 , 32 ], no three-dimensional (3D) structures are available for coronaviruses full-length N proteins. Here, by combining electron microscopy and biophysical experimental analysis with molecular modeling and molecular dynamics simulations, we propose structural models for the full-length SARS-CoV-2 N protein in the absence and presence of RNA. These models not only support current experimental data, but also provide a framework for understanding the multifunctional role of the N protein.

The NTD and CTD are connected by a central disordered serine and arginine-rich region, denoted as SR linker. This linker region is also proposed to play fundamental roles in protein oligomerization and function. Of note, the SR linker was shown to be modified by phosphorylation, which not only reduces the affinity of the protein for the RNA, but also drives a liquid phase separation of the N protein with the RNA and other virus proteins and host cell components [ 28 – 30 ]. Recently, SR-linker mutations have been associated with the emergence of the high-transmissible SARS-CoV-2 lineage B.1.1.7 [ 31 ]. Therefore, understanding how the NTD and CTD are oriented in structure and how the SR-linker and other unstructured regions contribute to the overall N protein organization is of paramount importance.

Coronaviruses N proteins are composed of two structured and globular domains represented by the N- (NTD) and C-terminal (CTD) domains, both of which are capable of binding single-stranded RNA and DNA molecules [ 15 – 24 ]. The NTD has an extensive basic U-shaped RNA-binding cleft implicated in the binding and melting of transcription regulatory sequences (TRS) needed for transcription of sub-genomic RNAs [ 9 , 14 ]. The CTD is responsible for the protein dimerization and it also forms a positively charged groove thought to contribute to the recognition of the packaging signal (PS) and to the assembly of the RNP into the virion particle [ 13 , 17 , 23 ]. In addition to the CTD, the flexible C-terminal tail also seems to influence protein oligomerization by promoting protein tetramerization [ 25 – 27 ]

N proteins are conserved among coronaviruses and are known to play multiple roles in the virus life cycle [ 4 ]. In addition to packaging the viral genomic RNA, N proteins are required for genome replication, transcription and translation, and for the assembly of the RNPs into newly formed viral particles [ 5 – 13 ]. This functional diversity is intimately linked to the dynamic structure of the N protein and its ability to bind and alter the RNA structure [ 9 , 14 ].

All coronaviruses, including the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of the Coronavirus disease 2019 (COVID-19) pandemics, possess an organized nucleocapsid formed by a ribonucleoprotein (RNP) complex surrounded by a lipid envelope [ 1 – 3 ]. The major component of the RNP complex is the nucleocapsid (N) protein, one of the four structural proteins of coronaviruses and also the most abundantly expressed viral protein in infected host cells [ 2 ].

Results

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[1] Url: https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1010121

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