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Dynamics of the N-terminal domain of SARS-CoV-2 nucleocapsid protein drives dsRNA melting in a counterintuitive tweezer-like mechanism

View ORCID ProfileÍcaro P. Caruso, View ORCID ProfileKaroline Sanches, View ORCID ProfileAndrea T. Da Poian, View ORCID ProfileAnderson S. Pinheiro, View ORCID ProfileFabio C. L. Almeida
doi: https://doi.org/10.1101/2020.08.24.264465
Ícaro P. Caruso
1Multiuser Center for Biomolecular Innovation (CMIB) and Department of Physics, Institute of Biosciences, Letters and Exact Sciences (IBILCE), São Paulo State University (UNESP), 15054-000, São José do Rio Preto, SP, Brazil
2Institute of Medical Biochemistry Leopoldo de Meis (IBqM) and National Center for Structural Biology and Bioimaging (CENABIO), Federal University of Rio de Janeiro (UFRJ), 21941-590, Rio de Janeiro, RJ, Brazil
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  • For correspondence: falmeida@bioqmed.ufrj.br icaro.caruso@unesp.br
Karoline Sanches
1Multiuser Center for Biomolecular Innovation (CMIB) and Department of Physics, Institute of Biosciences, Letters and Exact Sciences (IBILCE), São Paulo State University (UNESP), 15054-000, São José do Rio Preto, SP, Brazil
2Institute of Medical Biochemistry Leopoldo de Meis (IBqM) and National Center for Structural Biology and Bioimaging (CENABIO), Federal University of Rio de Janeiro (UFRJ), 21941-590, Rio de Janeiro, RJ, Brazil
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Andrea T. Da Poian
2Institute of Medical Biochemistry Leopoldo de Meis (IBqM) and National Center for Structural Biology and Bioimaging (CENABIO), Federal University of Rio de Janeiro (UFRJ), 21941-590, Rio de Janeiro, RJ, Brazil
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Anderson S. Pinheiro
3Department of Biochemistry, Institute of Chemistry, Federal University of Rio de Janeiro (UFRJ), 21941-590, Rio de Janeiro, RJ, Brazil
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Fabio C. L. Almeida
2Institute of Medical Biochemistry Leopoldo de Meis (IBqM) and National Center for Structural Biology and Bioimaging (CENABIO), Federal University of Rio de Janeiro (UFRJ), 21941-590, Rio de Janeiro, RJ, Brazil
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  • For correspondence: falmeida@bioqmed.ufrj.br icaro.caruso@unesp.br
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ABSTRACT

The N protein of betacoronaviruses is responsible for nucleocapsid assembly and other essential regulatory functions. Its N-terminal domain (NTD) interacts and melts the double-stranded transcriptional regulatory sequences (dsTRS), regulating the discontinuous subgenome transcription process. Here, we used molecular dynamics (MD) simulations to study the binding of SARS-CoV-2 N-NTD to non-specific (NS) and TRS dsRNAs. We probed dsRNAs’ Watson and Crick (WC) base-pairing over 25 replicas of 100 ns MD simulations, showing that only one N-NTD of dimeric N is enough to destabilize dsRNAs, initiating melting. N-NTD dsRNA destabilizing activity was more efficient for dsTRS than dsNS. N-NTD dynamics, especially a tweezer-like motion of β2-β3 and 2-β5 loops, played a key role in WC base-pairing destabilization. Based on experimental information available in the literature, we constructed kinetics models for N-NTD-mediated dsRNA melting. Our results support a 1:1 stoichiometry (N-NTD:dsRNA), matching MD simulations and raising different possibilities for N-NTD action: (i) two N-NTDs of dimeric N would act independently, increasing efficiency; (ii) two N-NTDs of dimeric N would bind to two different RNA sites, bridging distant regions of the genome; and (iii) monomeric N would be active, opening up the possibility of a regulatory dissociation event.

IMPORTANCE Coronaviruses are among the largest positive-sense RNA viruses. They display a unique discontinous transcription mechanism, involving N protein as a major player. The N-NTD promote the dsRNA melting releasing the nascent sense negative strand via a poorly known mechanism of action. It specifically recognizes the body TRS conserved RNA motif located at the 5’ end of each ORF. N protein has the ability to transfer the nascent RNA strand to the leader TRS. The mechanism is essential and one single mutation at the RNA binding site of the N-NTD impairs the viral replication. Here, we describe a counterintuitive mechanism of action of N-NTD based on molecular dynamics simulation and kinetic modelling of the experimental melting activity of N-NTD. This data impacts directly in the understanding of the way N protein acts in the cell and will guide future experiments.

Competing Interest Statement

The authors have declared no competing interest.

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Posted September 06, 2020.
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Dynamics of the N-terminal domain of SARS-CoV-2 nucleocapsid protein drives dsRNA melting in a counterintuitive tweezer-like mechanism
Ícaro P. Caruso, Karoline Sanches, Andrea T. Da Poian, Anderson S. Pinheiro, Fabio C. L. Almeida
bioRxiv 2020.08.24.264465; doi: https://doi.org/10.1101/2020.08.24.264465
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Dynamics of the N-terminal domain of SARS-CoV-2 nucleocapsid protein drives dsRNA melting in a counterintuitive tweezer-like mechanism
Ícaro P. Caruso, Karoline Sanches, Andrea T. Da Poian, Anderson S. Pinheiro, Fabio C. L. Almeida
bioRxiv 2020.08.24.264465; doi: https://doi.org/10.1101/2020.08.24.264465

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